Technology of Combined Identification of Macrophages and Collagen Fibers in Liver Samples.

The importance of identifying pathological changes in the liver both in fundamental researches and in diagnostic practice dictates the necessity to have a convenient method of assessing functional condition of resident macrophages and connective tissue fibers. The aim of the study is to assess the technology of combined histo-immunohistochemical detection of collagen fibers of connective tissue and resident liver macrophages using aniline blue histological stain and available antibodies to the microglial marker, the Iba-1 protein.

Materials and methods: Liver samples from adult rats (n=6) have been used in the study. The connective tissue was stained with a 2% aqueous solution of aniline blue (Unisource Chemicals Ltd., India). Monoclonal rabbit antibodies to Iba-1 (Clone JM36-62; ET1705-78; HuaBio, China) were used to detect resident liver macrophages, zinc-ethanol-formaldehyde was employed as a fixative.

Results: The combined staining method allowed us to detect numerous Iba-1-immunopositive structures corresponding morphologically to Kupffer cells and connective tissue macrophages, background staining was not observed. Staining with aniline blue in the liver samples was selective, uniform, and clear, and allowed for differentiation of the connective tissue in all examined samples. Exclusion of the heat-induced epitope retrieval stage caused no negative effect on identification of macrophages, reduced the probability of non-specific staining of the collagen fibers with aniline blue, and ensured general preservation of tinctorial properties of the liver tissue.

Conclusion: The presented protocol of combined histo-immunohistochemical identification of Kupffer cells and connective tissue fibers, applied on the rat liver samples, makes it possible to perform effectively the morphometric analysis and may find its application in pathohistological, clinical, and preclinical investigations.