[PDF] Immunohistochemistry (IHC-FR)-Frozen sections protocol

Discover more at abcam.com/technical Immunohistochemistry (IHC-FR)-Frozen sections protocol Frozen sections: Once mounted on APES coated slides, frozen sections are best kept at -80°C until needed. 1. When required, leave to warm at room temperature for 5 mins. 2. Pre cool the fixative (acetone, methanol or ethanol) at -20°C for 30 mins. (Abcam recommends starting with acetone) 3. Fix with the pre cooled fixative for 5-10 mins, at room temperature. 4. Rinse 3-4 X in PBS. 5. Continue with the immunohistochemical staining protocol. The absence of a formaldehyde based fixative eliminates the need for an antigen retrieval step. However, if frozen tissue or cytological specimens have been fixed in formalin, antigen retrieval can be attempted, although the friable nature of the specimens, in particular brain tissue, may compromise the success. The following reference describes a protocol in which slides are treated with 3-aminopropyltriethoxysilane (APES) before placing sections on the slides. This treatment improved adhesion, allowing heat mediated antigen retrieval with minimal damage to the tissue morphology: Warembourg M, Leroy D., Microwave pre treatment of sections to improve the immunocytochemical detection of progesterone receptors in the guinea pig hypothalamus. J Neurosci Methods. 2000 Dec 15;104(1):27-34.A more thorough discussion of antigen retrieval applied to frozen tissue sections is found in the following reference: Yamashita S, Okada Y. Application of heat-induced antigen retrieval to aldehyde fixed fresh frozen sections. J Histochem Cytochem. 2005 Nov;53(11):1421-32. If the tissue samples are fixed with an aldehyde fixative such as formalin, paraformaldehyde or glutaraldehyde and immunofluorescence (IF) is the detection method, consider including 0.3 M glycine in the blocking buffer, before applying the primary antibody. Glycine will bind free aldehyde groups that would otherwise bind the primary and secondary antibodies, leading to high background. Background due to free aldehyde groups is more likely to occur when the fixative is glutaraldehyde or paraformaldehyde. See IHC-Paraffin protocol (IHC-P) for detailed protocols for chromogenic and fluorescent detection.