Tissue fixation is critical for immunohistochemistry. Recently, we developed a zinc-ethanol-formalin fixative (ZEF), and the present study was aimed to assess the applicability of the ZEF for the human brain histology and immunohistochemistry and to evaluate the detectability of different antigens in the human brain fixed with ZEF. In total, 11 antigens were tested, including NeuN, neuron-specific enolase, GFAP, Iba-1, calbindin, calretinin, choline acetyltransferase, glutamic acid decarboxylase (GAD65), tyrosine hydroxylase, synaptophysin, and α-tubulin. The obtained data show that: i) the ZEF has potential for use in general histological practice, where detailed characterization of human brain morphology is needed; ii) the antigens tested are well-preserved in the human brain specimens fixed in the ZEF.

Mast cells play an important role in the body defense against allergens, pathogens, and parasites by participating in inflammation development. However, there is evidence for their contributing to the pathogenesis of a number of atopic, autoimmune, as well as cardiovascular, oncologic, neurologic, and other diseases (allergy, asthma, eczema, rhinitis, anaphylaxis, mastocytosis, multiple sclerosis, rheumatoid arthritis, inflammatory gastrointestinal and pulmonary diseases, migraine, etc.). The diagnosis of many diseases and the study of mast cell functions in health and disease require their identification; so, the knowledge on adequate imaging techniques for mast cells in humans and different species of animals is of particular importance.

The pineal gland plays a key role in coordinating various bodily functions. The majority of pineal cells are pinealocytes, and the second largest group are glial cells, the data on which are contradictory. The present work is undertaken to study astroglial cells of the human pineal gland using an immunohistochemical method with transmitted light microscopy and, for the first time, confocal laser microscopy. Astrocytes were labeled with antibodies to glial fibrillary acidic protein (GFAP) and vimentin. A large number of GFAP- and vimentin-immunopositive structures have been found in the human pineal gland. GFAP was localized in polygonal cells located in lobules among pinealocytes, while vimentin was localized in blood vessels and rounded cells localized mainly in trabeculae and partially in pineal lobules. Both GFAP- and vimentin-immunoreactive cells had several long branching processes that penetrated the entire pineal parenchyma, forming a dense network, and ended on the surface of the pineal gland, blood vessels, and around calcifications. GFAP-immunoreactive fibers tightly entwined all calcifications (singly and in groups), while vimentin-immunopositive processes surrounded only a part of them. The study of consecutive sections of the pineal gland showed that the coincidence of the localization of GFAP and vimentin for pineal cells is not typical. It can be supposed that, in the human pineal gland, there are two separate populations of astrocyte-like cells, GFAP- or vimentin-containing, which differ not only cytochemically, but also in morphological features and localization of cell bodies, as well as in the location of processes.

Objective: Immunohistochemical investigation of archival histological material is a serious problem, since long-term storage of biological tissues, most often in formalin, leads to a loss of antigenic properties. However, the biological material can also be stored in the clearing agent methyl salicylate. The aim of this study was to assess the antigenicity of the human choroid plexus after extra long-term storage in methyl salicylate. Material and Method: The study was performed on samples of fixed human choroid plexus (occasionally with attached neighboring pineal gland) stored in either methyl salicylate or paraffin blocks for 25 years. Chromogenic and fluorescence immunohistochemistry of vimentin, GFAP, type IV collagen, beta-catenin, alpha-smooth muscle actin, von Willebrand factor, CD68, mast cell tryptase, TMEM119, and synaptophysin was carried out. Results: The storage of human choroid plexus in methyl salicylate for 25 years does not impair its histomorphology and preserves the properties of all the antigens assessed, which makes their immunohistochemical visualization possible using both light and fluorescence microscopy. Additionally, we found that long-term storage of human choroid plexus in methyl salicylate does not cause an increase in autofluorescence. Conclusion: Methyl salicylate can be recommended as a medium for long-term storage of biological tissue, as it provides excellent brain tissue preservation and retains its antigenic properties for up to 25 years.