Abcam
Decant the secondary antibody solution and wash three times with PBS for 5 min each in the dark. Page 4. 4. ICC and IF protocol. Multicolor immunostaining. (
Abcam
Double immunofluorescence – simultaneous protocol. In order to be able to examine the co-distribution of two (or more) different antigens in the same sample
Immunocytochemistry (ICC) and immuofluorescence (IF)
Discover more at abcam.com In order to examine the co-distribution of two (or more) different antigens in the same sample a double immunofluorescence ...
IHC-PARAFFIN PROTOCOL (IHC-P)
Proper fixation is key for the success of immunohistochemistry.10% neutral buffered formalin (NBF) is most commonly used. Where Abcam's datasheets state IHC-P
ICC/IF of suspension cells using Cytospin™
Discover more at abcam.com. 1 of 2. ICC/IF of suspension cells using. Cytospin™. Immunocytochemistry/immunofluorescence (ICC/IF) is a technique that uses
Abcam
paraformaldehyde or glutaraldehyde and immunofluorescence (IF) is the detection See IHC-Paraffin protocol (IHC-P) for detailed protocols for chromogenic and.
Whole mount fluorescent immunohistochemistry
www.abcam.com/technical. Whole mount fluorescent immunohistochemistry. Procedure based on protocols and information kindly provided by: Professor Anthony
ab108410 – DRAQ5™ (5 mM) Protocol
4 июл. 2013 г. Protocol I: Immunofluorescence. 6. Protocol II: Flow Cytometry. 10. 6 ... www.abcam.com
Abcam
Caspase immunofluorescence staining protocol. Apoptosis is a highly regulated mechanism of cell death which converges on caspase activation. Immunofluorescence
FIXATION AND PERMEABILIZATION IN IHC/ICC
Acetone fixation will also permeabilize. Page 2. www.abcam.com/technical. 2. Methanol fixation can be used to permeablize but is not always suitable. These
Abcam
immunofluorescence protocol. Procedure for staining of cell cultures using immunofluorescence. Page 2. 2. ICC and IF protocol. Preparing the slide.
Abcam
Discover more at abcam.com/technical. Double immunofluorescence – simultaneous protocol. In order to be able to examine the co-distribution of two (or more)
IHC-PARAFFIN PROTOCOL (IHC-P)
www.abcam.com/technical. IHC-PARAFFIN PROTOCOL (IHC-P). Immunohistochemistry (or IHC) is a method for demonstrating the presence and location of proteins in
Immunocytochemistry (ICC) and immuofluorescence (IF)
Discover more at abcam.com. 2 of 3. Blocking and Immunostaining. 1. Incubate cells with 1% BSA in PBST for 30 min to block unspecific binding of the
FIXATION AND PERMEABILIZATION IN IHC/ICC
Acetone fixation will also permeabilize. Page 2. www.abcam.com/technical. 2. Methanol fixation can be used to permeablize but is not always suitable. These
Abcam
Immunostaining – free-floating sections. –. Signal amplification. Paraffin and frozen sections. Reagents can be applied manually by pipette or the protocol
Abcam
Discover more at abcam.com/technical. Stripping for reprobing The following two protocols differ in harshness of treatment. As a rule of thumb ...
Whole mount fluorescent immunohistochemistry
www.abcam.com/technical. Whole mount fluorescent immunohistochemistry. Procedure based on protocols and information kindly provided by:.
IMMUNOHISTOCHEMISTRY (IHC-FR) - Abcam
5. Continue with the immunohistochemical staining protocol. The absence of formalin eliminates the need for an antigen retrieval step. However if frozen tissue
Abcam
The following western blotting protocol includes the process of sample preparation gel electrophoresis
FIXATION AND PERMEABILIZATION IN IHC/ICC
FIXATION:
Fixation should immobilize antigens while retaining cellular and subcellular structure. It should also allow for access
of antibodies to all cells and subcellular compartments. The fixation and permeablisation method used will depend
on the sensitivity of the epitope and antibody themselves, and may require some optimization.Fixation can be done using crosslinking reagents, such as paraformaldehyde. These are better at preserving cell
structure, but may reduce the antigenicity of some cell components as the crosslinking will obstruct antibody
binding. For this reason, antigen retrieval techniques may be required, particularly if there is a long fixation
incubation or if a high percentage of crosslinking fixative is used. Another option is to use organic solvents. These
remove lipids while dehydrating the cells. They also precipitate proteins on the cellular architecture.
1. 4% Paraformaldehyde
Add 4% paraformaldehye to slides for 10 minutes only.Rinse with PBS or PBS 1% BSA
Note: Fixing in paraformaldehyde for more than 10-15 minutes will cross link the proteins to the point where antigen
retrieval may be required to ensure the antibody has free access to bind and detect the protein.2. Ethanol
Add 100-200ul per slide of cooled 95% ethanol, 5% glacial acetic acid for 5-10 minutes. Wash with PBS or PBS
1% BSA
3. Methanol
Add 100-200ul per slide of ice cold methanol.
Place at -20oC for 10 minutes.
Wash with PBS or PBS 1% BSA
Note: methanol will also permeabilize, but not in all cases as some epitopes are very sensitive to this. Can try
acetone instead for permeabilization if required.4. Acetone
Add 100-200ul per slide ice cold acetone. Place at -20oC for 5 to 10 minutes.Wash with PBS or PBS 1% BSA
Note: acetone will also permeabilize, no permeabilization step required.PERMEABILIZATION
Permeabilization should only be required for intracellular epitopes when the antibody required access to the inside
of the cell to detect the protein. However, it will also be required for detection of transmembrane membrane
proteins if the epitope is in the cytoplasmic region.Solvents:
1. Acetone fixation will also permeabilize
www.abcam.com/technical 2. Methanol fixation can be used to permeablize but is not always suitable.These reagents can be used to fix and permebilize, or can be used after fixation with a crosslinking agent such as
paraformaldehyde to permeabilize the cells.Detergents:
1. Triton or NP-40
Use 0.1 to 0.2% in PBS, 10 minutes only.
These will also partially dissolve the nuclear membrane and are therefore very suitable for nuclear antigen staining.
Note: as these are harsh detergents, they will disrupt proteins if they are used at higher concentrations or for longer
amounts of time which will affect staining results.2. Tween 20, Saponin, Digitonin and Leucoperm
Use 0.2 to 0.5% for 10 to 30 minutes.
These are much milder membrane solubilizers. They will give large enough pores for antibodies to go through
without dissolving plasma membrane. Suitable for antigens in the cytoplasm or the cytoplasmic face of the plasma
membrane. Also suitable for soluble nuclear antigens.SPECIAL RECOMMENDATIONS:
Cytoskeletal, viral and some enzyme antigens usually give optimal results when fixed with acetone, ethanol or
formaldehyde (high conc).Antigens in cytomplasmic organelles and granules will require a fixation and permeabilization method depending
on the antigen. The epitope needs to remain accessible.quotesdbs_dbs10.pdfusesText_16[PDF] aberdeen wa fireworks 2020
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