Negative-staining autoradiography: a new technique for ultracryotomy utilizing an interposed film

A new radiocytochemical technique is reported for ultrastructural localization of diffusible substances, using negatively stained ultra-cryostat sections. A sheet of film interposed between the cryostat section and the emulsion layer has rendered negative-staining autoradiography (NSA) practical. The rationale of NSA is that the film completely shields the section from all moisture-producing autoradiographic processes, so that phosphotungstic acid (PTA) can stain the section either before or after autoradiography (ARG), without the possibility of ultrastructural damage by alkaline solutions, interference between PTA and photoprocessing compounds, and superimposed images of a gelatin layer stained with PTA. As a model to demonstrate the newly developed procedure of NSA, rat brains were labeled with [125I]-triiodothyronine, fixed with tannic fixative, immersed in a cryoprotectant, frozen in liquefied propane, and cryostat sectioned. The resulting higher yield of radioactivity (85%) on the section was confirmed by a radiation counter. The retention rate was approximately 20% greater than that of conventional sections. Developed silver grains were found on synaptic vesicles and mitochondria in the polymorphic layer of the dentate gyrus. In this report we will also discuss the problems associated with cryostat sectioning of fresh tissues, the concept of ARG resolution, the distribution pattern of developed silver grains, and the possible applications of NSA.     

A new staining technique for microfilariae.

A rapid whole mount staining method is described to identify and differentiate microfilariae without elaborate processing. A single solution combining Hoyer's mounting medium and hematoxylin stain facilitates light microscopic examination of nuclei and sheaths of microfilariae. The new technique stains microfilariae adequately in three to seven minutes at 60--64 C making the method preferable to conventional methods that may take as long as 45 to 60 minutes. Lantern heat may be used to heat slides in rural areas with good results.     

An improved technique for rapid autoradiography of cells and tissue sections

Summary We have demonstrated a method for autoradiography of cell and tissue preparations which is both rapid and safe. The method utilizes only the primary scintillator, PPO, placed under the final emulsion to facilitate activation of the silver grains in the emulsion. Exposure of the autoradiographs is complete under the conditions described within 4 h at ambient temperature. The method is sensitive to exposure time and to the concentration of added radioisotope. The exclusion of volatile, toxic chemicals from the preparations allows the experiments to be performed without any health hazard to the investigator.     

A new technique for staining catecholic residues in biological samples

This technique for localizing catecholic residues in biological samples is based on the condensation of Besthorn's hydrazone (3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) with quinone residues obtained by the oxidation of catechols in the presence of ammonia. The product is a dark pink MBTH-quinone compound. This method is very sensitive and positive to catechol even at the 0.05 microgram level and the final product is chemically stable.     

A new technique for staining mast cells using ferroin

We describe here a new method for specific staining of mast cells using ferroin. Different hamster tissues were fixed in 4% formalin and processed for paraffin embedding. Sections were stained with hematoxylin followed by ferroin acidified with 2.5 N sulfuric acid to pH 4.0. Mast cells stained an intense orange color that contrasted markedly with bluish violet nuclei. High contrast was also observed when ferroin colored sections were counterstained with light green instead of hematoxylin. To evaluate the specificity of the stain, hamster cheek pouch sections were stained with toluidine blue, alcian blue-safranin O, and ferroin. Quantitative evaluation of mast cells stained with the three techniques showed no statistical difference. The simplicity and selectivity of this method is sufficient for image analysis of mast cells.     

A new technique for staining mast cells using ferroin

We describe here a new method for specific staining of mast cells using ferroin. Different hamster tissues were fixed in 4% formalin and processed for paraffin embedding. Sections were stained with hematoxylin followed by ferroin acidified with 2.5 N sulfuric acid to pH 4.0. Mast cells stained an intense orange color that contrasted markedly with bluish violet nuclei. High contrast was also observed when ferroin colored sections were counterstained with light green instead of hematoxylin. To evaluate the specificity of the stain, hamster cheek pouch sections were stained with toluidine blue, alcian blue-safranin O, and ferroin. Quantitative evaluation of mast cells stained with the three techniques showed no statistical difference. The simplicity and selectivity of this method is sufficient for image analysis of mast cells.     

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.