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Immunohistochemistry can be performed on tissue that has been fixed and embedded in paraffin, but also cryopreservated (frozen) tissue.Based on the way the tissue is preserved, there are different steps to prepare the tissue for immunohistochemistry, but the general method includes proper fixation, antigen retrieval incubation with primary antibody, then incubation with secondary antibody.
One of the main difficulties with IHC staining is overcoming specific or non-specific background. Optimisation of fixation methods and times, pre-treatment with blocking agents, incubating antibodies with high salt, and optimising post-antibody wash buffers and wash times are all important for obtaining high quality immunostaining.
The chemical composition and pH value of the buffer solution also contribute to the effectiveness of heat-induced antigen retrieval. [1] Thus, the AR-immunohistochemistry protocol must be optimized for each tissue type, fixation method, and antigen using a "test battery" to maximize antigen recovery in formalin fixed paraffin embedded sections. [1]
After blocking, a solution of primary antibody (generally between 0.5 and 5 micrograms/mL) diluted in either PBS or TBST wash buffer is incubated with the membrane under gentle agitation for typically an hour at room temperature, or overnight at 4°C. It can also be incubated at different temperatures, with lesser temperatures being associated ...
The newly transferred blots are then soaked in a blocking solution; non-fat milk and bovine serum albumin are common blocking buffers. [8] This blocking solution assists with preventing non-specific binding of the primary and/or secondary antibodies to the nitrocellulose membrane. Once the blocking solution has adequate contact time with the ...
A blocking antibody is an antibody that does not have a reaction when combined with an antigen, but prevents other antibodies from combining with that antigen. [1] This function of blocking antibodies has had a variety of clinical and experimental uses. The term can also be used for inhibiting antibody, prozone phenomenon and, agglutination ...
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