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Immunohistochemistry - Pretreatment

  • Fixation
  • Processing, Embedding and Microtomy

 Tissue fixation is used for several reasons, which include prevention of putrefaction from bacteria, autolysis from enzyme degradation and loss of soluble substances. Tissue fixation helps enhance staining in immunohistochemisty.

However it is important to have the tissue fixed as soon as possible and also to avoid over fixation, with a fixation time of between 4-24 hours being the most effective. This is also an issue with fixation for later antigen retrieval, as the formalin fixation process leads to the formation of protein cross links. This can mask their presence and requires pretreatment either with heat or enzymes to break these links before the process can be used. Further details on fixation can be found here

Tissue then undergoes processing, with dehyrdation in ethanol to remove water, then clearing through a suitable agent like xylene before the impregnation of the tissue using molten paraffin wax.  

This is the most common technique as the tissue is now in paraffin wax, which is very useful for tissue as it provides support for the tissue. Once impregnated this allows embedded to form a parrafin block, which allows sectioning with microtomy, while preserving tissue almost indefinately. Further details on tissue processing can be found here