A simple staining method for chromatin in electron microscopy compatible with serial sectioning

The study of the disposition of chromatin in the interphasic nucleus requires the combination of serial sectioning and a specific or preferential chromatin staining. The staining of chromatin with phosphotungstic acid (PTA) chromatin was originally employed on sections of glycolmethacrylate-embedded samples. As it is very difficult to obtain ribbons with this resin, we introduced a modification which consisted of staining the tissue after fixation and before dehydration, in order that epoxy resins can be applied. Several procedures were tried and the best results were attained in the following way. Standard fixation of samples no thicker than 1 mm was carried out with 2.5% glutaraldehyde at pH 7.2 for 1 or 2 h at room temperature; tissues were then rinsed three times with 0.2N HCl adjusted to pH 2.1-2.3 with 0.2N NaOH for 15 min. Staining was held with 3% W/V PTA in 1N HCl adjusted to the same pH. Samples were dehydrated in gradual ethyl alcohol concentrations and Epon-embedded. Post-staining on sections with uranyl-acetate and lead-citrate or other methods may be used to demonstrate other cell components and their relations to chromatin.