Combination of diaminobenzidine staining and immunogold labeling: a novel technical approach to identify lysozyme in human neutrophil cells.
In this study, a combination of the diaminobenzidine staining procedure for myeloperoxidase and the immunogold labeling technique was successfully used to show that lysozyme is indeed found in both the primary and secondary type granules of human neutrophils. Following the systematic selection of processing conditions by light microscopic peroxidase anti-peroxidase cytochemistry, on slide preparations, consistent gold labeling was obtained over both types of granules. The combination of myeloperoxidase and immunogold cytochemical procedures permitted the lysozyme-labeling pattern of the small-sized granules to be studied in isolation, thereby confirming the existence of lysozyme in secondary granules. In addition, myeloperoxidase was observed in the large-sized, lysozyme-positive, granules by both cytochemical and immunocytochemical methods, thereby confirming that these labeled structures were primary granules. Morphometrical analysis confirmed that there was a significant difference in mean size between the lysozyme-positive, myeloperoxidase-positive, granules and the lysozyme-positive, myeloperoxidase-negative, granules. The former were significantly larger in size than the latter. In conclusion, although the localization of lysozyme in human neutrophils by the immunogold technique is confirmatory, the combination of enzyme cytochemistry and immunocytochemistry is a novel technical approach that permits the lysozyme-labeling patterns of granule types to be studied in isolation. This double labeling technique is relatively straightforward and, as such, consistent immunostaining can be routinely obtained using intact cells.
Mutasa
H C
HC
Department of Haematology, Cambridge University Clinical School, United Kingdom.
D015100:3,3'-Diaminobenzidine; D003594:Cytoplasmic Granules / Q000201:enzymology*; D005404:Fixatives; D006801:Humans; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D009113:Muramidase / Q000032:analysis*; D009504:Neutrophils / Q000201:enzymology* / Q000648:ultrastructure; D009195:Peroxidase / Q000032:analysis; D013194:Staining and Labeling
D005404:Fixatives; D015100:3,3'-Diaminobenzidine; D009195:Peroxidase; D009113:Muramidase