Preservation of the structural integrity of a freshly lesioned or transplanted mouse neocortex and the immunoreactivity of cell-specific marker proteins in demineralized histological material

• Published in: Journal of neuroscience methods 1995-11, Vol.62 (1), p.55-63
• Main Authors: Ourednik, Jitka, Ourednik, Wenzel
• Format: Article
• Language: English
• Subjects: Animals; Antibody Specificity; Biomarkers - analysis; Calbindin; Calbindins; Central nervous system injury; Cerebral Cortex - cytology; Cerebral Cortex - surgery; Cerebral Cortex - transplantation; Demineralization; Glial fibrillary acidic protein; Glial Fibrillary Acidic Protein - analysis; Immunohistochemistry - methods; Mice; Mice, Inbred C57BL; Microtubule-associated protein 2; Microtubule-Associated Proteins - analysis; Minerals - analysis; Nerve Regeneration - physiology; Neural graft; Neuroglia - chemistry; Neuroglia - cytology; Neuroglia - immunology; Neurons - chemistry; Neurons - cytology; Neurons - immunology; Regeneration; S100 Calcium Binding Protein G - analysis; Specimen Handling; Thy-1; Thy-1 Antigens - analysis
• ISSN: 0165-0270; 1872-678X
• DOI: 10.1016/0165-0270(95)00054-2

Analysis of superficially lesioned or grafted brains poses the problem that their removal from the skull prior to histological processing often causes damage to the operated area and may lead to loss of the graft. Here, we propose an original approach to this problem, developed on mice whose cortices have been surgically lesioned and some grafted with fetal neural tissue. The experimental animals were killed 1, 3 or 6 days after operation. Our procedure was based on the softening of skulls by demineralization in Gendre's picric solution, followed by solidification of the wounded region by embedding in polyester wax. This permitted the preparation of serial sections from brains together with the neurocrania. To check their immunoreactivity, the sections were later reacted with specific antisera for glial fibrillary acidic protein (GFAP), microtubule-associated protein 2 (MAP2), calbindin, and the thermolabile cell-surface glycoprotein Thy-1. The histological material revealed excellent structural integrity and cytoarchitecture. In transplanted animals, the tiny graft, protected by the overlying bone, was found in the host cavity. Immunostaining showed typical localization of the chosen marker proteins. The anti-Thy-1 antibody enabled us to distinguish between graft and host tissues, which differed, in our experiments, in their expression of two distinct allelic forms of the Thy-1 molecule. The method lends itself perfectly to histochemical study of the earliest stages of freshly operated superficial brain regions in small laboratory animals, and should also be applicable to the evaluation of other brain structures which are difficult to gain access to without being damaged.