Introduction to Cell & Molecular Biology Techniques

32185_7MolBiolWS08Immunofluorescence.pdf

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Introduction to Cell & Molecular Biology

Techniques :

Immunofluorescence

DAPI (DNA) GFP ɶ Tubulin

Merge

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SPARQed is a collaboration between The Uniǀersity of Yueensland͛s Diamantina Institute and The Yueensland

Government͛s Department of Education, Training and Employment.

The Immunofluorescence Workshop is based on technique used in the SPARQ-ed Research Immersion

Programs developed by scientists at the University of Queensland Diamantina Institute. The experimental

protocols and all supporting materials were adapted for student use by Dr Peter Darben, under the supervision

of Dr Sandrine Roy.

Cover Image : A field of HeLa cells stained with DAPI (which binds to DNA and fluoresces blue) and treated with

an antibody against ɶ Tubulin and a secondary antibody conjugated to a red flurophore. Some of these cells

have been transfected with the gene for the jellyfish protein green fluorescent protein (GFP). The figure shows

the three colour channels, imaged separately for blue, green and red, and the combined image obtained when

these three channels are merged. Image courtesy Rose Boutros.

All materials in the manual are Copyright 2014, State of Queensland (Department of Education and Training).

Permission is granted for use in schools and other educational contexts. Permission for use and reproduction

should be requested by contacting Peter Darben at p.darben@uq.edu.au. These materials may not be used for

commercial purposes

Risk assessments were developed with the assistance of Paul Kristensen, Maria Somodevilla-Torres and Jane

Easson.

Many thanks to all in the ͞Gab Lab" whose patience and support made this happen.

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Table of Contents

Introduction

What is Cell Biology ? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Common Cell Biology Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

The Workshop

Getting Started . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Theoretical Basis of the Project

Tissue Culture and Cancer Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Preparation of Cells for Immunofluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Antibodies and Immunofluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fluorescence Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Experimental Protocols

How to Use this Manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Preparation of Culture Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Blocking and Permeablisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Treatment with Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Imaging Using Epifluorescence Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

What to Look Out For . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Appendices

Appendix A : DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Appendix B : Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Appendix C : Using a Micropipette . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Appendix D : Glossary of Terms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4 4 6 6 7 9 13 14 19 19 19 20 21
21
22
24
29
34

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Introduction

What is Cell Biology ?

Cells form the basis of all living things. They are the smallest single unit of life, from the simplest bacteria to

blue whales and giant redwood trees. Differences in the structure of cells and the way that they carry out their

internal mechanisms form the basis of the first major divisions of life, into the three kingdoms of Archaea

(͞ancient" bacteria), Eubacteria (͞modern" bacteria) and Eukaryota (eǀerything else, including us). An

understanding of cells is therefore vital in any understanding of life itself.

Cell biology is the study of cells and how they function, from the subcellular processes which keep them

functioning, to the way that cells interact with other cells. Whilst molecular biology concentrates largely on the

molecules of life (largely the nucleic acids and proteins), cell biology concerns itself with how these molecules

are used by the cell to survive, reproduce and carry out normal cell functions.

In biomedical research, cell biology is used to find out more about how cells normally work, and how

disturbances in this normal function can result in disease. An understanding of these processes can lead to

therapies which work by targeting the abnormal function.

Common Cell Biology Techniques

The following list covers some of the more commonly used cell biology techniques - it is by no means

exhaustive.

Cell / Tissue Culture - in the same way that bacteria and other simple organisms can be grown in the

laboratory outside their normal environment, cells and tissues from more complicated organisms can

be cultured as well. The techniques are slightly different, and the culture media are more complex to

reflect the complex internal environment inside the host from which the cells are derived, however

cell and tissue culture is a powerful tool which provides an almost limitless supply of test material for

researchers to use without resorting to using whole organisms. In addition, the controlled conditions

in cell and tissue culture allow researchers to carry out experiments with a lower number of variables

which may affect the outcome of the test. Cell culture may use cells removed directly from an

organism (primary culture), or it may use lines of cultured cancer cells. The benefit of the latter

approach is that cancer cells continue to divide, while primary cultures cease dividing after a number

of cycles.

Microscopy - the basic tool of cell biology is microscopy. Recent advances in imaging technology have

allowed an unprecedented amount of information to be gleaned from microscopic analysis. Types of microscopic techniques which are used include : Brightfield - traditional microscopy, where cells are illuminated by visible light. Brightfield

microscopy gives a general picture of cell function, although that information is not very

detailed or specific. As animal cells lack cell walls, brightfield microscopy may use special

techniques such as phase contrast to show cellular structures in more detail. Brightfield

microscopy allows imaging of live or fixed (dead) cells and tissues)

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Electron Microscopy - uses a focused beam of electrons instead of light. Electron microscopy

permits a much higher magnification of specimens than light microscopy and is useful in

obtaining detailed information about sub-cellular structures. Electron microscopy requires extensive processing and so can only be performed on fixed specimens. Transmission electron microscopy provides a cross section of a specimen, while scanning electron microscopy gives a three-dimensional image of the surface of a specimen. Fluorescence Microscopy - uses fluorescent materials to indicate structures in a specimen.

Fluorescence occurs when light of one waǀelength ͞edžcites" a material and causes it to emit

light of a different wavelength. Most fluorescent materials give off visible light after excitation by ultraviolet light. Structures may be naturally fluorescent (autofluorescence) or they may be labeled with a compound which is fluorescent (eg. DAPI is a dye which binds onto DNA. The DNA and nuclei of cells stained with DAPI emit a blue light under ultraviolet light). Immunofluorescence - antibodies are proteins made by the immune system which bind onto specific parts of proteins. Antibodies can be raised against any protein in the cell. If these antibodies are attached to a fluorescent tag, the tag will only show up where that antibody attached (ie. where the target protein is found in the cell). Immunofluorescence allows very specific targeting of cellular structures. RNA Interference - RNA interference uses short sequences of RNA which are complementary to the

mRNA which carries to instructions to translate proteins from the DNA to the ribosomes. The

interfering RNA binds to the target sequence, preventing it from being translated. As a result, careful

selection of interfering RNA can be used to silence a particular gene. This allows researchers to study

what role a protein plays in a cell, by observing what happens when that protein is absent.

Timelapse Microscopy - many cellular processes (eg. mitosis) occur over a period of time which is not

practical for direct observation. Imaging cells over a period of time (eg. a photograph is taken every 20

minutes for 24 hours) allows us to combine these images in a ͞moǀie" which compresses a long time

period into a shorter one.

The Workshop

Whilst the nucleus and some other organelles are easily visualized using brightfield microscopy, most other

sub-cellular structures cannot be differentiated or even seen using conventional microscopes. Therefore these

structures and biomolecules must be demonstrated using more specific techniques such as immunofluorescence and fluorescence microscopy.

In this project you will be provided with a coverslip containing cancer cells grown in culture. You will then

expose the cells to antibodies directed against proteins found in subcellular structures, allowing these primary

antibodies to bind to those structures. You will then treat the cells with secondary antibodies directed against

the primary antibodies. These secondary antibodies have been conjugated to a fluorescent dye, effectively

attaching a fluorescent label to the structure you are interested in seeing. You will then use a fluorescence

microscope to image the cells, taking images of each colour channel and then combining the images to create

a merged image which demonstrates all structures.

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Getting Started

Before you begin, make sure that you are familiar with the relevant theory behind the techniques we will be

performing. This manual contains several appendices which will provide you with this information. Make sure

you read this information before proceeding.

Appendix A : DNA

Appendix B : Proteins

Appendix A : Using a Micropipette

Appendix B : Glossary of Terms

Information on other cell and molecular biology concepts and techniques are provided at the SPARQ-ed

website at : http://www.di.uq.edu.au/sparqedservices

Theoretical Basis of the Project

Tissue Culture and Cancer Cell Lines

When researchers want to obtain sufficient quantities of organisms to use in their studies, they may grow

them up in a vat of broth containing all of the nutrients they need to survive. This is called culturing the

organisms, and for many simple, free-living organisms such as bacteria or fungi, the process is relatively

straightforward. These organisms often require very little in the way of specialised nutrients and so long as the

culture conditions are suitable, they will undergo almost constant division, increasing their populations at an

exponential rate.

If cells from a multi-cellular organism like a human are to be used in research, the culture methods are not

nearly so simple. Firstly, cells from multi-cellular organisms have differentiated to such an extent that they can

no longer survive without the complex systems of nutrients and stimuli provided by the other cells in the body.

As a result, growing human cells in culture requires the use of growth media containing a complex mixture of

basic nutrients and specific growth factors provided by the inclusion of serum (the liquid component of clotted

blood). In addition, body cells spend most of their time carrying out the functions which allow them to play

their roles within the body. This means that they are not constantly dividing - in many cases they must be

stimulated to enter mitosis, and in some cases do not divide at all. As a result, human cells in culture often

proliferate very slowly.

One solution to this problem is the use of cancer cells. One of the hallmarks of cancer is the loss of regulation

of the cell cycle, leading to cells constantly passing through division cycles. This results in hyperproliferation of

the cells, which in the body leads to the growth of tissue masses known as tumours. Cancer cell

hyperproliferation means that they can be grown in culture for many generations, with further cultures able to

be sub-cultured from the original - they are effectively immortal. This has led to the production of cancer cell

lines, each of which was derived from a tumour recovered from an individual with cancer.

Most types of cancer have numerous cell lines which researchers can use to study the cancer in question. Each

of these cell lines have particular characteristics dependent on the cancer from which they were derived,

allowing researchers to select the line which most closely matches the situation they are working on.

Unfortunately, being cancer cells, the cells contain errors and abnormalities which mean that they do not

often behave as normal cells do, making them inappropriate models for normal cells, or even for cancers of

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other parts of the body. In addition, the same errors which remove the cell cycle controls in cancer cells may

also remove mechanisms which regulate damage to the DNA, allowing mutations to accumulate in the cells

which make them even more distinct from the cells from which they were derived HeLa cells are possibly the best known of all cancer cell lines. They were originally recovered from a cervical cancer removed from a patient named Henrietta Lacks in 1951. The cancer resulted from an infection by a strain of human papillomavirus (HPV18), and so the genome of these cells also contains the genome for this strain of HPV. HeLa was the first cell line to be successfully and continuously cultured in vitro and have been used widely around the world since the physician who first subcultured them made them and the techniques used to grow them freely available to scientists around the world. HeLa cells have played an important role in many important medical discoveries, including the development of the Polio vaccine. In this workshop, you will be using a genetically modified strain of HeLa cells used by the Cell Cycle Research Group at the University of Queensland Diamantina Institute. These cells have a modified gene for one of the histone proteins in the nucleus (H2), where the gene includes a gene derived from a jellyfish (the gene for green

fluorescent protein, or GFP). The histones are a family of proteins around which DNA wraps and which assist in

the packaging of DNA and help to regulate the expression of genes. When the modified gene is expressed, the

protein produced includes a GFP ͞tag" on the end which glows green when edžposed to blue light. This has

produced a cell line in which the nucleus fluoresces green, which means that this green signal can be used in

place of a DNA dye such as DAPI. This fluorescence is produced in living cells, allowing them to be used to

study the function and appearance of the nucleus at different stages of the cell cycle through live cell imaging.

Preparation of Cells for Immunofluorescence

Using routine brightfield microscopy, a reasonable amount of detail in the cell can be determined. Large

structures such as the nucleus (and its nucleoli) and vacuoles are easily distinguished inside the cell with even

fairly rudimentary microscopes. Some other structures can also be demonstrated with the careful use of dyes

and stains which give a colour based on chemical reactions with cellular components, and this forms the basis

of cytochemistry (on individual cells) and histochemistry (on thin sections of tissue). However, even these

methods are not nearly specific enough to properly demonstrate the wide variety of subcellular structures and

materials. Immunofluorescence is a technique which uses the highly specific binding of antibodies to their

target antigens as a way of demonstrating materials and structures inside cells.

Before any immunofluorescence procedures can be carried out on the cells, they must be properly prepared.

For this workshop, the cells have been grown on a coverslip placed on the bottom of a culture plate. When a

new culture is needed, a suspension of cells is prepared with the cells floating around inside the cell culture

medium. The number of cells per unit volume (generally per millilitre of medium) can be calculated using cell

counters and a specific number of cells added to a dish containing culture medium by adding the correct

volume of cell suspension (eg. if the density of cells in the suspension is 100,000 cells / mL and you needed

Henrietta Lacks

The patient from

whom the HeLa cell line was derived was Henrietta Lacks, who was living in

Baltimore in the US

at the time of her diagnosis. While the contribution that the HeLa cell line made is well known, it is an interesting case of medical ethics, as permission for the use of her tissue was never sought. More information about the life of Ms Lacks and the issues around the use of her cells can be found in the book The Immortal

Life of Henrietta Lacks by Rebecca Skloot.

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100,000 cells to start a culture, you would add 1mL of suspension). The exact seeding density depends on the

reproductive rate of the cells you are using and how dense you want the culture to be in the time you have.

When the cells are added to the culture dish, they sink to the bottom and attach through proteins embedded

in the cell membrane. Cells that have attached to the bottom of the dish take on the appearance of a fried egg,

with the nucleus as the yolk and the cytoplasm as the white. Cells only release from the bottom of the flask

when they are in the process of dividing, becoming spherical as they carry out mitosis. For this workshop, the

cells have been added to a 6-well culture plate. In each well, a number of coverslips were added to the wells

prior to the addition of the cells. When the cells settled to the bottom of the well, some landed on the

coverslip. This means that the cells can be easily removed from the well and treated while on the coverslip,

and eventually the coverslip containing the cells can be mounted onto a microscope slide for observation. The

coverslips have been previously treated with Poly-L-Lysine, a peptide which acts as a cellular ͞glue" and assists

in holding the cells (including mitotic cells which have rounded up) onto the coverslip.

At several times during the course of the immunofluorescence procedures, cells will need to be washed. This is

usually done by replacing the liquid in the well they are contained in with phosphate buffered isotonic saline, a

solution of sodium chloride at 9g/L in a buffer which keeps the pH at a physiological level of around 7.2.

Because the cells are stuck to the coverslip, the liquid can be gently removed and replaced with a solution

which does not put the cells under osmotic stress.

Once cells die (which starts to occur once their nutrients and ideal growing conditions have been removed),

they start to break down and lose their structure and integrity. A typical live cell taken through the processes

needed for immunofluorescence would have started the breakdown process by the time the techniques have

been completed. Therefore the cells must be preserved prior to undergoing antibody treatment. This process

is called fixation, and is the same technique used to preserve biological specimens in museums. Fixation relies

on ͞freezing" the proteins in place using a chemical process rather than temperature. The tertiary structure of

proteins is maintained by weak hydrogen bonds between the side chains of the amino acids that make up the

proteins. These hydrogen bonds are easily broken by increases in temperature or changes in pH, resulting in a

loss of protein structure called denaturation. Formalin is a buffered solution of the gas formaldehyde which

forms permanent covalent crosslinks between protein chains and locks them into their tertiary structure,

regardless of minor changes in temperature or pH, and thus preserves the structure of the cell. In this

investigation, you will use a type of formalin called paraformaldehyde to fix the cells, although other fixatives

like ice cold methanol can also be used.

Once cells have been washed, fixed in paraformaldehyde and washed again, they must then be permeablised.

The antibodies used for immunofluorescence are large protein molecules which cannot cross the cell

membrane, so the cell membrane must be removed. It is very important that the cells must be fixed prior to

permeablisation, otherwise the cell will burst.

To understand why it is important to fix the cells, it may help to imagine the cell as a dome tent filled with

cooked spaghetti (with the nucleus as a beachball inside it, perhaps). The shape of the cell is maintained by

structural proteins such as Tubulin, represented in our analogy by the cooked spaghetti, while the whole shape

of the cell is maintained by the cell membrane, represented by the fabric of the tent itself. If you were to cut

away the tent fabric (removing the cell membrane through permeablisation), all of the spaghetti would fall out

and spill onto the ground. Washing the cell would then have the effect of hosing the mess away.

Fixing the cells would be like drying the spaghetti out. It would shrink slightly and revert back to its state prior

to cooking, however it would be in the same position it was in when the drying occurred. The tent fabric (cell

membrane) could then be removed and you would be left with a mound of dried spaghetti in the same shape

as the tent, but without the tent around it (see Figure 1). Chemical fixation does make some changes to the

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proteins in the cell, howeǀer enough of the original structure is often left to retain some of the proteins͛

functions, including the ability of the proteins to bind antibodies and the fluorescence characteristics of green

fluorescent protein (both of which are vital for this investigation.

In this workshop, cells will be permeablised using a detergent (Triton X100). Detergents work by solublising

lipids in water, and since the cell membrane is made up of lipid molecules, the Triton X100 in this method

removes the lipids from the membrane, allowing large molecules such as antibodies to enter the cell and bind

to proteins inside. At the same time as they are permeablised, the cells will be blocked. Blocking involves the

addition of a solution of proteins (usually bovine serum albumin, or BSA). Cells may contain proteins which can

bind onto antibodies and other proteins non-specifically, rather than the antibodies specifically binding onto

their target proteins. The BSA ͞soaks up" these non-specific binding sites, ensuring that the only way the antibodies can bind is via their specific targets. Once permeablised, blocked and washed once more, the cells are ready to be treated with antibodies.

Antibodies and Immunofluorescence

Antibodies are large, complex proteins made by specific immune cells in the body (the plasma cells, which are derived from populations of B lymphocytes). Antibodies are produced in response to the presence of an agent in the body which the immune system recognizes as being not from the body. These agents are usually foreign materials, such as proteins on the surface of microbes, although in some cases the immune system may target the body͛s own proteins, causing autoimmune diseases such as Type I

Diabetes and Rheumatoid Arthritis.

Permeablisation

Unfixed Cell

Permeablisation Fixed Cell

Figure 1 - Fate of unfixed and fixed cells following permeablisation

Variable Regions

Light Chain

Heavy Chain

Figure 2 Generalised Antibody Structure

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Antibodies are composed of four peptide chains (two light chains and two heavy chains) arranged like a capital

letter ͞Y" (see Figure 2). Most of an antibody͛s structure is constant between antibodies (with some ǀariation

between different classes), howeǀer at the ends of the ͞arms" of the Y is a small, highly ǀariable region made

up of a region of the light and heavy chains. It is changes in the shape of this region which determines the

difference between antibodies. The shape of this variable region is such that it fits around and binds very

specifically to a region on the material it is raised against. The area which it binds to is called the epitope, while

the material where the epitope is found is called the antigen. A different antibody is made in response to each

epitope on each antigen, with the variable regions confirming exactly to each epitope. This means that

antibodies are made which bind very specifically to particular epitopes on antigens. This specificity is so high

that two antibodies can be generated which can tell the difference between two versions of the same protein,

one which has a phosphate group attached and one which does not. This high level of specificity makes

antibodies extremely useful in detecting particular proteins, and is used in techniques such as diagnosis of

disease agents in the blood, western blotting, immunohistochemistry, and, of course, immunofluorescence.

Antibodies are made in animals, or in cell lines derived from those animals. To generate an antibody against a

particular protein, samples of that protein are injected into a test animal. A better antibody response is

generated if the target protein comes from a different species to the animal used to raise the antibody as the

host animal would recognize the protein as being foreign. For example, if you are interested in using

antibodies to detect a human protein, you would use a non-human animal such as a mouse, rabbit or goat to

raise the antibody. Once the animal starts making the antibody, it can be recoǀered from the animal͛s blood

and purified for use. In some cases the lines of plasma cells which are making the antibody can be isolated and

fused with a cancer cell, making a hybridoma cell line which has the immortality of the cancer cells and the

antibody-generating properties of the plasma cells. This means that the antibodies can be produced in culture

without the need for using further animals.

The technique you will be using is indirect immunofluorescence. Direct immunofluorescence involves

chemically combining a fluorescent dye directly to the primary antibody raised against the protein you are

interested in. The antibody, with its fluorescent label binds, onto the target protein in the cell, essentially

colouring the target protein with the label (see Figure 3). Direct immunofluorescence is not often used now, as

it would involve making a range of differently labeled antibodies for every protein studied in laboratories. In

addition, the sensitivity of the technique is low, as there is only one label for each binding site.

With indirect immunofluorescence, the primary antibody is left unlabelled. It still binds to the target protein,

however it must be itself demonstrated using a labeled secondary antibody. This secondary antibody is

generated in the same way as the primary antibody, however its target is antibodies from the species in which

the primary antibody has been raised. For example, if the primary antibody was raised in a mouse, the labeled

secondary antibody would be raised in another species against mouse antibodies (eg. goat anti-mouse). This

allows laboratories to have a wide selection of primary antibodies directed against any protein they might be

working on, and then a smaller selection of labeled secondary antibodies directed against the species the

primary antibodies were raised in (eg. anti-mouse, anti-rat, anti-goat, anti-chicken, etc). In addition, because

multiple labeled secondary antibodies can bind to a single primary antibody, the signal strength is higher,

resulting in greater sensitivity (see Figure 4).

If primary antibodies from different species are used, then multiple proteins can be targeted in the one cell.

The only limits are the number of species available and the range of dyes used. For example, a mouse derived

primary antibody against protein A could be used alongside a goat derived primary antibody against protein B,

so long as the anti-mouse and anti-goat secondary antibodies had different coloured labels.

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Antigen Absent Antigen Present

Unbound antibody washed away

Antigen Absent Antigen Present

Treatment with labeled antibody

Fluorescence observed where antigen is located

UV Light

Antigen Present Antigen Absent

UV Light

Figure 3 - Principal of Direct Immunofluorescence

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Antigen Absent

Unbound antibody washed away

Antigen Present

Treatment with unlabelled primary antibody

Antigen Present Antigen Absent

Treatment with unlabelled secondary antibody

Antigen Present Antigen Absent

(continued over) Figure 4 - Principle of Indirect Immunofluorescence

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The general protocol for antibody treatments is to expose the cells to the primary antibody for 1-2 hours, wash

thoroughly in PBS to remove all unbound primary antibody, then expose to the secondary antibody for 1 hour.

The cells are washed in PBS to removed any unbound secondary antibody, and washed in water to remove the

PBS (which forms beautiful, but annoying crystals when the coverslips are mounted). To make a permanent

preparation, the coverslips are mounted on a microscope slide in a special mounting medium which hardens

upon contact with air.

Targets

In this workshop, you will be demonstrating three components of the cells :

The Nucleus - the HeLa H2B GFP cell line expresses a green fluorescent protein tag on the H2 Histone

protein located in the nucleus. This protein is found throughout the nucleus and remains associated with

the DNA, even during mitosis. These cells therefore show a green fluorescence in the nucleus, and in the

chromosomes during mitosis.

Unbound secondary antibody washed away

Antigen Present Antigen Absent

Fluorescence observed wherever antigen is located

Antigen Present Antigen Absent

UV Light UV Light

Figure 4 - Principle of Indirect Immunofluorescence (Continued)

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ɲ-Tubulin (Alpha Tubulin) - ɲ-Tubulin is one of the subunits of the tubulin protein which forms an

important part of the cytoskeleton which provides support to the cell. During interphase, tubulin is

found throughout the cytoplasm of the cell right up to the cell membrane and has a fibrous appearance.

During mitosis, the cell assembles the mitotic spindle from Tubulin, and this structure appears as a

͞birdcage"-like structure on which the chromosomes are arranged. Tubulin is also a major component of

the centrioles which make up the centrosome, and this appears as paired pinpoint bodies within the

cytoplasm of the cell. In this inǀestigation, the antibody against ɲ-Tubulin used will be derived from a

rabbit, and a secondary anti-rabbit antibody conjugated to a far-red (647nm) secondary antibody will be

used to demonstrate it. The human eye cannot detect light at 647nm easily, so a false colour (eg.

magenta) will be applied, and the ɲ-Tubulin will appear as fine fibres of this colour throughout the

cytoplasm. Mitotic spindles will also be demonstrated using this method. Complex IV (or Cytochrome C Oxidase) - Complex IV is a large transmembrane protein composed of 13

subunits and found in the mitochondria of eukaryotic cells. Complex IV catalyses the last step in the

electron transport chain in cellular respiration, transferring electrons to molecular oxygen and allowing

the production of water molecules. In eukaryotic cells, apart from a couple of exceptions, Complex IV is

found only in the membranes of mitochondria, which means that this protein complex can be used as a

mitochondrial ͞marker" t a target which demonstrates the presence of mitochondria in the cell. The

antibody against Complex IV used in this workshop is derived from a mouse, and a secondary anti- mouse antibody conjugated to a blue (405nm) fluorescent label will be used to demonstrate it. The mitochondria should appear as blue specks throughout the cytoplasm of the cell.

Fluorescence Microscopy

In routine light microscopy, visible light is passed through a specimen on a microscope slide. This light

continues up through the optics of the microscope to the eyes. Structures in a specimen will interfere slightly

with the passage of the light, absorbing some wavelengths to give it a colour, or subtly bending it to reveal the

shape of the structure. The structures visible through light microscopy may be enhanced through the use of

coloured dyes which bind to components in the specimen, which is the basis of histochemistry, a useful

technique to visualize elements in normally colourless animal tissue.

In fluorescence microscopy, structures are visualized using fluorescent dyes as labels. Fluoresence is a

phenomenon where the chemical structure of the dye captures electromagnetic radiation of one wavelength

(the excitation wavelength) and releases it as radiation of another, lower energy wavelength (the emission

wavelength). For example, when light at 488nm (in the blue region of the visible spectrum) falls upon green

fluorescent protein, electrons in the outer orbital of the atoms within the protein are excited to a higher

energy state. When they return to their normal energy state, they emit photons of light at 509nm, which is in

the green region of the visible spectrum (see Figure 5).

In fluorescence microscopy, light of the desired excitation wavelength is shone onto the specimen, exciting the

fluorescent labels. The light emitted is passed up through the optics of the microscope to the eyes or a light

detector. To prevent interference from reflected excitation light entering the optics, a dichroic mirror is used,

which allows the emitted light to pass through, but not the excitation light. Further sensitivity can be achieved

using emission filters, which only allows light of the desired emission wavelength to pass through. When light

is shone down through the entire specimen, this is known as epifluorescence. Fluorescence microscopes have

a number of adjustable filters, so that a range of excitation and emission wavelengths can be selected (see

Figure 6).

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e- GFP

488nm

e- GFP

509nm

Figure 5 - Excitation of GFP by light at 488nm (blue) to create emission at 509nm (green) Figure 6 - Schematic of an Epifluorescence Microscope

Specimen

Objective

Lens

Emission Filter

Eyepiece lens

White Light

Excitation

Filter

Dichroic

Mirror

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Due to the arrangement of excitation and emission filters, fluorescence microscopes can only image a single

fluorophore at a time. Therefore, in specimens which have multiple fluorophores, each with different

excitation and emission wavelengths, multiple images must be taken. The microscope is set up for each

fluorophore and an image taken before it is set up for the next fluorophore. The collected images, called

channels, are then combined to create the final image. The light emitted by the fluorophores is often very

weak, or at a wavelength which is difficult to detect with the eyes (the 647nm fluorophore used in this

investigation, for example, cannot be seen by the human eye). Therefore, the digital cameras used in

fluorescence microscopes take the images in grey scale as full colour detectors are less sensitive. Before the

channels are combined, each is assigned a false colour which corresponds to the colour of the light emitted

(see Figure 7).

Blue Channel

(DAPI/DNA)

Green Channel

(GFP)

Red Channel

(Tubulin)

False colour

Blue

False colour

Green

False colour

Red

Merge

Figure 7 - Combining channels to make a three-colour image

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Retaining the three channels in the final image can be useful for scientists, as the grayscale images make it

easier to see finer detail than the colour ones, and comparing separate channels makes it easier to detect

different fluorophores co-localised or situated close to one another. As a result, when presenting their results

in lectures or scientific papers, scientists often present the three channels as grayscale images, followed by the

combined colour image (see Figure 8).

A limitation of using epifluorescence microscopy is the difficulty in determining whether a combined signal is

the result of two fluorophores in contact with each other (co-localisation). For example, if a green flurophore

and a red fluorophore appear close to each other in the combined image, they appear yellow. This could be

due to the structures they are attached to being in close physical contact, an important consideration when

using fluorescence microscopy to determine interactions between proteins. However cells are three

dimensional objects with a reasonably large depth of field, so the two fluorophores may not actually be close

to each other, but actually overlapping through the depth of the cell (see Figure 9).

One solution to this problem is the use of confocal microscopy. In this method, background fluorescence is

limited by the use of a pinhole aperture (at the expense of signal intensity, resulting in longer exposure times).

In addition, the excitation of the fluorophores is done by tightly focused lasers which scan through the

specimen. Therefore, only a particular portion of the specimen is exposed to the excitation wavelength and a

virtual section is created. If structures are co-localised, they will appear on the same section, whereas if they

are distant but overlapping, only one will appear on the section. Another benefit to confocal microscopy is that

seƋuential sections can be stacked on top of one another to create a three dimensional image (a ͞Z stack").

Due to the lack of background fluorescence, confocal images appear clearer than epifluorescence images (see

Figure 10).

Green

(GFP) Red (Tubulin) Blue (pPLK1) Merge Figure 8 - Demonstration of presentation of fluorescence microscopy results

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Co-localisation

Combined signal

appears yellow

Distant but

overlapping

Overlapping signal

appears yellow Figure 9 - Co-Localisation and overlapping signals a) b) Figure 10 - Comparison of epifluorescence and confocal microscopy.

a) HeLa cells imaged using epifluorescence - Blue is DAPI (staining DNA), red is tubulin, green is GFP

b) HeLa H2B GFP cells imaged using confocal microscopy - Blue is pPLK1, red is tubulin, green is GFP

(localized to nucleus)

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Experimental Protocols

How to Use this Manual

Throughout this section you will see a series of icons which represent what you should do at each point. These

icons are: Write down a result or perform a calculation.

Prepare a reaction tube.

Incubate your samples.

When you are asked to deliver a set volume, the text will be given a colour representing the colour of the

micropipette used: e.g. 750µL Use the blue P1000 micropipette (200-1000µL) 100µL Use the strong yellow P200 micropipette (20-200µL)
15µL Use the pale yellow P20 micropipette (2-20µL) 2µL Use the orange P2 micropipette (0.1-2µL)

Preparation of Culture Plates

Prior to this laboratory session cells have been seeded into 6 well culture plates with poly-L-lysine coated

coverslips on the bottom of each well. The cells have been allowed to attach to the coverslips and grow

overnight. The culture medium was removed from each well and the cells were washed three times by

replacing the medium with phosphate buffer saline (PBS - a solution which does not put the cells under

osmotic stress) and removing. The cells were then fixed in a solution of 4% paraformaldehyde in PBS for 20

minutes, before being washed three times again in PBS.

You will be working in pairs. Three pairs will work with one culture plate, allowing for two coverslips per pair.

Blocking and Permeablisation

Add 3mL of Block / Permeablisation solution (1X PBS containing 1% BSA and 0.1% Triton X100) to each of the remaining wells

6 Incubate the coverslips at room temperature for 15 minutes

# 6

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Treatment with Antibodies

The primary antibodies need to be diluted first in Blocking Solution (1XPBS containing 1% BSA). Prepare the

following antibody dilutions : 1° Antibody Source Volume

Needed

Volume

Block. Soln.

ɲ Tubulin Rabbit 1µL 500µL ɲ Compledž IV Mouse 1µL

In order to account for background fluorescence, a negative control should be prepared for the primary

antibodies. This coverslip should be placed on a drop of blocking solution rather than diluted antibody.

Set up 50µL spots of diluted primary antibody on a piece of Parafilm, one for each coverslip, as well as

a 50µL drop of blocking solution for the negative control Place each coverslip CELL SIDE DOWN on separate spots on the Parafilm

6 Incubate the coverslips under a foil cover for 1 hour at room temperature

Set up three 100µL spots of PBS for each coverslip on a separate piece of Parafilm Wash the coverslips by placing each CELL SIDE DOWN on each spot and transferring to the next Prepare the secondary antibody solutions by diluting them in Blocking Solution (PBS containing 1%

BSA) as indicated below :

Coverslip 2° Antibody Volume

Needed

Volume

Block. Soln.

ɲ Tubulin ɲ Rabbit 647 1µL

300µL

ɲ Compledž IV ɲ Mouse 405 1µL

Set up 50µL spots of the secondary antibody solutions on Parafilm and place each coverslip CELL SIDE

DOWN on separate spots. In this case, the negative control coverslip should be placed on a spot of diluted secondary antibodies

6 Incubate the coverslips under aluminium foil for one hour at room temperature

Wash the coverslips three times in PBS as before Rinse off the PBS by dipping the coverslip in water Prepare 10µL drops of Mowiol mounting medium on microscope slides

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Mount the coverslips CELL SIDE DOWN on the drops of Mowiol Place the slides under a stream of hot air from a floor heater for 15 minutes.

Imaging Using Epifluorescence Microscopy

Observe the coverslips using the fluorescence microscope as directed by you tutor. Most of the cells visible

should be in interphase, allowing you to see a normal nucleus and distribution of tubulin and mitochondria.

Due to the poly-L-lysine coating on the microscope, you should see some cells at different stages of mitosis.

The three components visualized should be :

The Nucleus / Chromosomes - The nucleus will appear as a pale green blob with a distinct boundary around its outside. Inside the nucleus should be some unstained regions which resemble bubbles -

these are the nucleoli and represent centres of RNA synthesis. During mitosis, the chromosomes

condense and the green signal will become stronger. In prophase and prometaphase, you will see the DNA clump together in bright green bundles (the chromosomes) while the clear boundary around the

outside of the nucleus will disappear. In cells in metaphase, the chromosomes line up as a line across

the middle of the mitotic spindle (the ͞metaphase plate"), while in anaphase and telophase, you should see the chromosomes separate out into separate bundles at opposite ends of the cell.

Tubulin - tubulin should appear in interphase cells as a diffuse fibrous red material. In mitosis, from

the end of prometaphase, this consolidates into the mitotic spindle - a birdcage like structure with

strands of tubulin radiating out from the ends. The mitotic spindle should start to break down by the

end of telophase.

Complex IV / Mitochondria - mitochondria should as tiny blue specks throughout the cytoplasm of the

cell - they are actually much smaller than how they are portrayed in diagrams in textbooks.

Things to Look Out For

The Stages of Mitosis - see if you can get an image of cells at each stage of mitosis

Aberrant Cells - HeLa cells have been continuously subcultured since 1952. This means that they have

accumulated a lot of mutations, on top of those that led to the cells turning cancerous in the first

place. Observe the diversity of cell shape and size in the cells present, along with other anomalies such

as cells with more than one nucleus. Aberrant Mitosis - some of the mutations HeLas have accumulated include changes to the normal process of mitosis. A more common anomaly is cells dividing in three rather than two - these cells have a three-pronged metaphase plate (like the Mercedes-Benz symbol) and in anaphase you will see the chromosomes being pulled in three directions rather than two. Where are the Mitochondria ? - what happens to the mitochondria during mitosis ? How are they parceled out between the two daughter cells ?

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Appendix A : DNA

Deoxyribonucleic acid (DNA) is a large molecule which stores the genetic information in organisms. It is

composed of two strands, arranged in a double helix form. Each strand is composed of a chain of molecules called nucleotides, composed of a phosphate group, a five carbon sugar (pentose) called deoxyribose and one of four different nitrogen containing bases. Figure 3 - The Structure of a Single Strand of DNA Each nucleotide is connected to the next by way of covalent bonding between the phosphate group of

one nucleotide and the third carbon in the deodžyribose ring. This giǀes the DNA strand a ͞direction" t

from the 5͛ (͞fiǀe prime") end to the 3͛ (͞three prime") end. By conǀention, a DNA seƋuence is always