A rapid phosphotungstic acid staining method on ultra-thin sections

Abstract

The Nail-Patella Syndrome (NPS) is an autosomal dominant hereditary disease in which about 60% of the patients have glomerular abnormalities. Phosphotungstic acid (PTA) has been used successfully to demonstrate the specific collagen fibrils of NPS within the glomerular basement membrane and/or mesangial matrix in ultra-thin sections. However, in our laboratory we had difficulty staining Eponate 12 sections with PTA. A modified staining procedure which makes use of the microwave has been developed as follows:1. Glutaraldehyde/osmium fixed kidney specimens were embedded in Eponate 12 and ultra-thin sections collected on Gilder 400 mesh copper grids. Grids were stained using a RTV 700 silicone rubber staining pad. This pad contains shallow, round wells (5mm in diameter and 2mm deep) with slots in the center bottom of each well. By bending the pad, the rim of a grid can be inserted into the slot. On release, the grids adhere securely to the wall of the slot.2. 10% aqueous phosphotungstic acid was dropped into the staining wells around each grid. The staining pad was then placed in a moisture chamber consisting of a pre-heated Petri dish containing wetted filter paper and placed in Pelco 3400 laboratory microwave oven for 110 seconds at 80% power. A Tripour beaker containing 200ml room temperature tap water was placed in the microwave oven as a heat sink.