OPTIMIZING COLLAGEN ANTIGEN UNMASKING IN PARAFFIN-EMBEDDED TISSUES

In order to optimize collagen antigen unmasking in paraffin-embedded t issue sections, the effects of various fixatives and duration of fixat ion in relation to enzyme pretreatment and microwave irradiation for c ollagen antigen unmasking were studied. A streptavidin-biotin-peroxida se complex method was used for the immunolocalization of type III and IV collagen antigens. Fixatives and fixation time had significant adve rse effects on the immunoreactivity of the antigens Enzyme pretreatmen t was found to be superior to microwave irradiation for collagen antig en unmasking. Fixation with paraformaldehyde required shorter enzyme p retreatment and yielded a more enhanced reaction than treatment with f ormalin and Bouin's fluid. The optimum conditions for type III and IV collagen unmasking were fecund to be fixation with 4% paraformaldehyde in 0.01 M phosphate-buffered saline, pH 7.4, for up to 3 weeks follow ed by enzyme pretreatment with 1 mg ml(-1) pepsin in 0.01 N hydrochlor ic acid, pH 2.0, for 30 min (human tissues) or 60 min (rat tissues) at 37 degrees C. It is concluded that collagen antigen unmasking by enzy me pretreatment in tissue sections fixed for a long period of time can be successful if appropriate enzyme's and incubation time(s) are empl oyed with regard to the antigen under study and fixative and fixation time used for tissue preparation.