Effects of heat fixation on tissue structure, immunostaining and in situ RT-PCR

The effects of PBS heat fixation on tissue structure, immunostaining, and in situ RT-PCR were investigated using excised mucosal tissues from both humans and various animals. It was found that basal cells were stretched to two to three times their normal length at 60 degrees C, and 4 to 5 times at 90 degrees C, resulting in detachment of the epithelium and basal cells from subjacent connective tissue. Tonofibrils disappeared completely and became an electron dense amorphous mass at 60 degrees C. Both nuclear and cytoplasmic membranes also disappeared as did intracytoplasmic organelles. Electron dense amorphous-material occupied the entire nucleus with fixation at 90 degrees C. Collagen fibrils appeared swollen at 60 degrees C and either disappeared completely or became fragmented undergoing transition to an electron dense amorphous conglomerates at 90 degrees C. Immunostaining of cytokines disappeared at 60 degrees C, and that of vimentin at 90 degrees C. However, cytokeratin and human leukocyte common antigen (CD 45RB) reactivity remained intact at both temperatures. IL-6 mRNA could be localized by in situ RT-PCR even after thermal fixation. Results suggest that a proper combination of immunohistochemistry and in situ RT-PCR will enable investigators to localize proteins in addition to DNA or RNA within oral tissue sections. This capability should prove helpful in applied histopathologic diagnostic techniques.