A method for the use of Zenker-formol fixation and the periodic acid Schiff staining technique in light microscopic radioautography

Summary

Although providing superior histological preservation, Zenker-formol fixation has not been utilized in radioautographic experiments, since some component of the fixative (presumably mercury) desensitizes the emulsion and thus prevents the appearance of a radioautographic reaction. Attempts to identify and eliminate the effects of this desensitizing agent led to experiments with Zenker-formol fixed tissues from rats injected with ³H-leucine or ³H-thymidine. Radioautographs of such tissues contained a brown-black precipitate and occasionally a heavy background fog, but no normal radioautographic reaction. Moreover, when the radioautographs were exposed to light, the emulsion was transparent over the tissues, indicating that a tissue-bound component had completely desensitized the emulsion. The two components of Zenker's fluid-mercuric chloride and potassium dichromate-plus the mercurous chloride which forms as a precipitate in the course of fixation, were then tested for their individual effects on the radioautographic emulsion. It was found that mercuric chloride was primarily responsible for the desensitization of the emulsion. Merourous chloride, which formed the dark precipitate in radioautographs, produced the background fog. While potassium dichromate caused some desensitization of the emulsion, in normal histological processing where tissues had a lengthy post-fixation wash, this substance was for the most part washed out.

A number of attempts were made to remove the mercury by treating the Zenker-fixed tissues with solutions of iodine, sodium thiosulfate and cysteine. It was found that the most intense radioautographic reaction was obtained with the following procedure: prior to embedding, tissue blocks were treated with 0.5% iodine solution in 70% alcohol for 17 hours. Optionally, sections of this material could be given a second treatment with 0.5% alcoholic iodine for 10 minutes. Although simple iodination eliminated the mercurous chloride precipitate and the background fog, there was still no radioautographic reaction. However, subsequent treatment of sections of this iodinated material in 5% aqueous sodium thiosulfate for 5 minutes yielded an adequate radioautographic reaction. Therefore, the simultaneous removal of both mercurous chloride precipitate and tissue bound mercury required iodination followed by sodium thiosulfate. Using this procedure, a method was described for preparing tissues for radioautography using Zenker-formol fixation and the periodic acid-Schiff staining technique.