PubMed · 1468782
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N B Friedman. 1992. On formalin fixation.. https://doi.org/10.1016/0046-8177(92)90070-j
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This chapter explores the combination of a nonradioactive in situ hybridization technique to detect mRNA with an imunnohistochemical labeling technique for use in formalin-fixed, paraffin-embedded, or frozen tissue sections. This technique allows the combination of detecting mRNA by in situ hybridization with immunohistochemical detection of a protein product or a cell surface marker without using any radioactive procedures. This technique is ideal for use on tissue sections when the aim is to identify which cells are producing a secreted protein product, such as a cytokine.
Fixation of tissues with formalin results in well-preserved morphology but to a high degree leads to degradation of nucleic acids, which substantially constricts the spectrum of applicable molecular techniques. The novel HOPE-fixative with subsequent paraffin embedding, as an alternative to formalin, has been shown to result in a morphological preservation comparable to formalin-fixed, paraffin-embedded specimens. Due to a similar workflow like in formalin-fixation and paraffin embedding, the HOPE technique can be successfully established within any pathological institute. We have shown that DNA, RNA and proteins are protected in HOPE-fixed, paraffin-embedded tissues for at least eight years. Moreover, we described procedures which permit successful application of all common molecular techniques such as in situ hybridization targeting either DNA or RNA, immunohistochemistry without antigen retrieval and for formalin-refractory antigens, PCR, RT-PCR, Western blot, Northern blot, and transcription microarrays to HOPE-fixed, paraffin-embedded tissues. Furthermore, HOPE-fixed tissues can be used for the construction of tissue microarrays for enhanced high-throughput analyses on the molecular level. Using the HOPE technique as its crucial methodological base, ex vivo model systems could be established, e.g. for the simulation of early events in human infections and detection of chemotherapy resistances in human cancer. In addition to tissues, cell-culture preparations have been prepared utilizing the HOPE technique, which were then successfully applied to in situ hybridization targeting mRNA or immunocytochemistry with excellent preservation of morphological details. Taken together, the HOPE technique to date represents an alternative fixation that is, in contrary to other procedures, scientifically broadly analyzed. Therefore new possibilities are opened up especially within the rapidly growing field of molecular pathology.