Triple staining of brain tissue for neuronal classification.

The described triple staining (PAP, aldehydefuchsin, and cresyl violet) enables the distinction of different neuronal types with regard to their content of neurotransmitters or neuropeptides and their lipofuscin pigmentation respectively. The use of filters improves the contrast of immunohistochemical and lipofuscin staining, and facilitates the differentiation of neurons under the microscope. An additional important point is the advantage that perikaria can be measured in the usual morphometric manner.     

In toto staining and preservation of peripheral nervous tissue

A simple quantitative modification of the in toto staining technique for nervous networks of Sihler is described. The results are demonstrated on the innervation pattern of the hard palate of the rat. Formalin fixed hard palates of rat were macerated and bleached in an aqueous solution of 3% potassium hydroxide with a few drops of 3% hydrogen peroxide added. Thereafter, the specimens were decalcified in Sihler's solution I (1 part glacial acetic acid, 1 part pure glycerin and 6 parts 1% chloral hydrate), the process being controlled by radiography. The specimens were next stained in Sihler's solution II (1 part Ehrlich's hematoxylin, 1 part pure glycerin and 6 parts 1% chloral hydrate). After staining, the non-nervous tissues were destained in Sihler's solution I. Destaining was checked microscopically and was stopped before the finest branches of the nerves began to fade. The specimens were then washed in a weak aqueous solution of lithium carbonate and cleared in increasing concentrations of glycerin. Good visualization of nervous structures and a deep field of observation resulted; orientation of the peripheral nerves with respect to surrounding structures was readily seen and a three-dimensional image of the nervous networks was obtained.     

Deplasticizing of thick Epon sections involving a new adhesive technique and staining of nervous tissue

The present communication deals with a technique developed for the selective staining of neural tissue in thick (10 micron) Epon sections. A new adhesive method was needed, because the known techniques are only applicable to 0.5-2 micron thin sections. The critical step in the procedure is the adhesion of the sections onto the slides. This is accomplished by heating the sections on top of a uniform layer of albumin glycerol on the slide followed by coating with celloidin. The results after deplasticizing and coagulation with this technique are comparable to those obtained by paraffin or frozen section techniques, but in addition have the advantage of Epoxy resin embedding e.g. the possibility of cutting undecalcified hard tissues and sections for serial reconstruction.     

Light and electron microscopic localization of ATPase in normal and degenerating testes of Syrian hamsters.

The distribution of Mg++-activated ATPase was determined with light and electron microscopy in normal and degenerating seminferous tubules. In the normal animals ATPase was localized in the interface between spermatids and Sertoli cells, in association with the cytoplasmic filaments contained within Sertoli cell processes, and in the lymphatic endothelium. ATPase activity increased in degenerating tubules as observed by light microscopy. Electron microscopic investigations of the degenerating tubules which contained only spermatogonia and Sertoli cells revealed reaction product on the outer surface of the Sertoli cell processes and within the interface between adjacent Sertoli cells. Reactaction product was also observed in the Sertoli cell processes between the cytoplasmic filaments and the cell membrane. Where filaments were absent in Sertoli cell processes, no reaction product was observed. These electron microscopic studies indicate that the increase in ATPase activity in testicular degeneration is probably a relative increase due to a loss of the germinal elements of the tubular epithelium and subsequent apposition of the Sertoli cell processes. We speculate that the ATPase activity localized within the Sertoli cell processes may be involved in providing an energy source for filament motility.     

Apolipoprotein D expression absence in degenerating neurons of human central nervous system

Apolipoprotein D (apo D), a lipocalin transporter of small hydrophobic molecules could play an important role in several neurodegenerative diseases. However, its role in those diseases remains unclear. There has been reported increments of apo D in relation with different neuropathologic diseases. Recently, we reported the absence of apo D in neurons of substantia nigra which can contribute to the lability of neurons to oxidative damage. In order to determine the relationship between apo D expression and neuronal death, we studied the expression of apo D in various regions of human brains from patients without any neurological or psychological disorders, in relation with the neuronal damage revealed by Fluoro-Jade B staining. The absence of expression for apo D in injured neurons and the negative staining for Fluoro-Jade B of neurons that express apo D was observed in all sections studied. These findings are in accordance with the role possibly played by apo D in the neuroprotection of the nervous system.