PARAFORMALDEHYDE FIXATION OF NEUTROPHILS FOR IMMUNOLABELING OF GRANULE ANTIGENS IN CRYOULTRASECTIONS

Paraformaldehyde (PFA) fixation was optimized to facilitate the immobilization and labeling of multiple granule antigens, using short fixation regimens and cryoultramicrotomy of unembedded neutrophils (PMNs). In the optimal protocol, extraction of azurophil granule antigens (especially of the abundant elastase) was obviated by manipulating the polymeric state of PFA, and hence its rate of cross-linking, by altering its concentration and pH in a multistep process, Primary fixation conditions used (4% PFA, pH 8.0, 5 min) favor fixative penetration and rapid cross-linking, Stable crosslinking of the antigen was achieved in a secondary fixation step using conditions that favor larger, more cross-linking polymeric forms of PFA (8% PFA, pH 7.2, 15 min), Immobilization of granule antigens was enhanced by flotation of cut sections on fixative (8% PFA, pH 8.0) before labeling and by using post-labeling fixation with 1% glutaraldehyde. The optimized protocol facilitated immobilization and immunolabeling of elastase, myeloperoxidase, lactoferrin, and cathepsin D in highly hydrated, unembedded PMNs.