A few notes on fixation
The usual fixative for paraffin embedded tissues is neutral buffered formalin(NBF). This equivalent to 4% paraformaldehyde in a buffered solution plus a preservative (methanol) which prevents the conversion of formaldehyde to formic acid. Because of the preservative, NBF has a shelf life of months, whereas 4% PF must be made fresh. Optimal histology requires adequate fixation, about 45 hrs at room temperature for thinly sliced tissue. Inadequately fixed tissue will become dehydrated during tissue processing, resulting in hard and brittle specimens. Alcohol based fixatives generally do not give good morphology but may be useful in special cases (such as BrdU staining). A particular challenge for the histopathology is immunostaining fixed specimens. In many cases formaldehyde fixation will prevent recognition of epitopes by the primary antibody. Occasionally, “antigen retrieval” procedures will improve results but usually frozen sections are a better bet. An alternative approach, suitable for thin or porous tissues, is to perform immunohistochemistry on fresh tissues and then post-fix and embed the tissue in paraffin.