The effects of progressive formaldehyde fixation on the preservation of tissue antigens

A wide variety of fresh post-mortem tissues was fixed in 4% formaldehyde for 6 hours, 1, 3, 7, 14 and 30 days to simulate possible fixation schedules in the diagnostic laboratory, before processing through alcohol and chloroform. The immunoreactivity of paraffin-embedded sections to 25 monoclonal and polyclonal antibodies commonly used in tissue diagnosis was tested employing an avidin-biotin-peroxidase technique. There was a distinct fall-off in the staining of many antigens including the lymphocyte antigens LN1, LN2, LN3 and UCHL1 after 3 days of fixation and the intermediate filament proteins vimentin, desmin and neurofilaments failed to be labelled by monoclonal antibodies after 1 day and only variable staining for the cytokeratins was retained. S100 protein, prostate specific antigen, thyroglobulin and carcinoembryonic antigen were more resilient and showed weak staining after 14 days exposure to formaldehyde. Trypsinization improved the staining for cytokeratins, neurofilaments, desmin and Factor VIII-related protein, but for other antigens, the immunoreaction was weaker and background staining was increased. For the effective application of immunohistochemical staining as a diagnostic tool, the duration of formaldehyde fixation should be kept to a minimum and enzyme digestion should only be judiciously employed for selected antigens.