Fixatives paraformaldehyde-based

Performing immunohistochemistry on these rehydrated paraffin sections frequently leads to poor results. In contrast, the same antibodies on paraformaldehyde-fixed and cryostat sections will give good results. The issue with formalin-fixed and paraffin-embedded tissue is that the exposure to formalin and dehydration alters the epitopes in the tissue. As a result, formalin-fixed and paraffin-embedded tissues need additional processing methods, known as epitope retrieval or antigen retrieval. Done before immunohistochemistry, epitope retrieval involves heating the sections in buffer with either an acid or base to allow the antibody to recognize the epitope. Also, the exact process of epitope retrieval can be different for individual antibodies. There are numerous papers and books on epitope retrieval and how to apply the method. 

The carbodiimide-(EDCDI)-based fixatives should be freshly made. The preservation of tissue fixed in buffered EDCDI alone is poor Thus, one can either perform a post fixation in 4% buffered paraformaldehyde (Section 2 1., item 5), or fix the tissue in a mixture of paraformaldehyde and EDCDI. Fixation should be overnight (16 h) at 4-8 C. 

The first step for a successful in situ hybridization is the fixation of the tissue. This will ensure target nucleic acid retention and preservation of the tissue morphology. Either crosslinking or precipitative fixatives can be used, and a preference for either of the two types of fixative has often been based on the different types of system under investigation (1-7). For hybridization of regulatory peptide mRNA, 4% paraformaldehyde appears to be the most effective, both on tissue blocks and on tissue culture preparations. 

Fix dissected adult tissues or embryos in fresh 4% paraformaldehyde/0.2% glutaraldehyde in 0.1 M phosphate buffer. Determine the time required by the size of the tissue to be fixed based on penetration rates of 1 mm/h at room temperature. (Perfusion can also be carried out if desired, see section 1.3.2.2),... 

Ultimately, the choice of fixatives is today restricted to those based on aldehydes that offer a good compromise between their cross-linking capabilities and rapidity of cell/tissue penetration. Such an outcome is also intimately connected with the development of effective heath-induced epitope retrieval (HIER) protocols that allow effective unmasking of a wide number of antigens (epitopes) in paraformaldehyde- (and other aldehyde-) fixed tissues see below). 

One of the two protocols presented here has been adjusted to process paraformaldehyde-fixed tissues while the second for tissues that require glutaraldehyde fixation since the primary antibodies utilized are developed with immunogens obtained by coupling small molecules (e.g., neurotransmitters) with carrier proteins (for details see AO). The techniques described herein are based on those... 

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