R&D Systems provides monoclonal, polyclonal and biotinylated antibodies for immunohistochemical use. The following protocol has been developed and optimized by R& D Systems” Immunohistochemical Laboratory. R&D Systems” antigen affinity-purified polyclonal and monoclonal antibodies have been used to stain frozen cells and tissues, as well as paraffin-embedded tissues. Immunohistochemistry protocols may require modification depending on the type of tissue used. Each investigator s hould determine the optimal conditions and working dilutions of antibodies. If using R&D Systems” primary antibodies, refer to the specific product insert to obtain an approximate working dilution. For all other reagents, follow the manufacturer”s instructions. For Research Use Only.

　　Primary AntibodiesUnlabeled or biotinylated antigen affinity-purified polyclonal antibodies (R&D Systems” AF or BAF series) or selected unlabeled or biotinylated monoclonal antibodies (R&D Systems” MAB or BAM series)

　　Cell & Tissue Staining Kits – Enzymatic/Chromogenic

　　Note: Equivalent chemicals and reagents may be substituted for those listed above.

　　The vast majority of immunohistochemical procedures employ a cell or tissue fixation step using formaldehyde or other cross-linking fixatives prior to incubation with primary antibody. Fixation is required to retain tissue morphology and prevent degradation of tissue antigens. Fixation may be performe d either by immersing dissected pieces of tissue (e.g. human biopsies) into the fixative, or by vascular perfusion (e.g. laboratory animals such as mice, rats, guinea pigs, etc.). It is very important to optimize fixing conditions since under- or over-fixation may reduce or abolish tissue immunoreactivity. The easiest way to co rrect under-fixation is to post-fix tissue sections on the slide before starting immunohistochemical staining. To recover antigens in over-fixed tissue s, either protease-induced epitope retrieval (PIER) or heat-induced epitope retrieval (HIER) techniques are recommended. HIER can be performed using a microwave oven, pressure cooker, vegetable steamer, autoclave or water bath. After tissues are fixed, they may either be embedded into paraffin or covered with OCT compound and frozen for further sectioning. Paraffin-embedded tissues are cut using a mi crotome at room temperature, whereas frozen tissues are cut using a cryostat at temperatures below 0° C. Antigen immunoreactivity was found to be better preserved in frozen rather than paraffin-embedded tissues.1,2

　　When it is not possible to fix tissue by perfusion, dissected tissue may be fixed by immersing the tissue into a 10 formalin solution for 4 – 8 hours at room temperature. It is commonly accepted that th e volume of fixative should be 50 times greater than the size of the immersed tissue. Avoid fixing the tissue for greater than 24 hours since tissue an tigens may either be destroyed or masked (A.C. Cuello, ed., 1993, Immunohistochemistry: Methods in the Neurosciences, Vol. 14; IBRO Handbook Series, John Wiley & Sons, New York).

　　Preparation of gel-coated slides

　　Gel Coating Solution:

　　Gel-coated Slides:

　　Antigen-retrieval ProtocolThis protocol is based on using R&D Systems” Antigen Retrieval Reagents (Catalog # CTS013, CTS014, CTS015 or CTS016). Note: the antigen-retrieval capacity of each Antigen Retrieval Reagent depends on sample preparation, antigen structure, incubation time (up to 30 minutes) and temperature (90 – 100° C). Each investigator should determine optimal conditions.