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Formalin fixed tissue requires an antigen retrieval step before immunohistochemical staining can proceed. This is due to the formation of methylene bridges during fixation, which cross link proteins and therefore mask antigenic sites. The two methods of antigen retrieval are either enzymatic or heat mediated. Both serve to break the methylene bridges and so expose the antigenic sites in order to allow the antibodies to bind. Some antigens prefer the enzymatic method to heat mediated antigen retrieval and vice versa. The enzymatic method tends to be a much gentler process than heat mediated method, so is best suited to more sensitive tissues. However, the enzymatic method tends to take much longer and is more technically demanding.If no antigen retrieval step is stated on the antibody data sheet, start off by trying the heat mediated method. If at first you don't succeed, try again using the enzymatic method.Frozen tissue sections do not need an antigen retrieval step. Once mounted on APES coated slides, they are best kept at -80°C until needed. When required, allow the slides to warm at room temperature for 5 minutes, then acetone fix for 5 minutes followed by a PBS or TBS rinse. Afterwards, continue with the immunohistochemical staining protocol.
Tissue sections are best mounted on APES (amino-propyl-tri-ethoxy-silane) coated slides. Slides should be placed in a standard rack for this procedure.
The use of a domestic microwave is inadvisable. Hot and cold spots are common which lead to uneven antigen retrieval. In addition, antigen retrieval times are usually longer, due to the absence of a pressurized environment that nearly always leads to section dissociation.A scientific microwave is much more appropriate. Most brands have on-board pressurized vessels and can keep the temperature at a constant 98 degrees C to avoid section dissociation. The only drawback is the expense of purchasing one! But if your department has one by all means use it.Tissue sections are best mounted on APES (amino-propyl-tri-ethoxy-silane) coated slides. Slides should be placed in a standard plastic rack for this procedure.
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