Reproducibility of periodic acid–methenamine silver staining: Its application resulting in color tone changes progressing to the renal tubular brush border and chronological changes in methenamine silver reaction

<p>Periodic acid–methenamine silver (PAM) staining is often used for the histopathological examination of kidney tissues. However, this technique has low reproducibility, especially with thick sections, as the silver reaction is difficult to control. Therefore, we attempted to improve the staining technique by changing the sequence of the methenamine silver reaction, the actual conditions, and the endpoint of the method. We then examined the effects of these changes on staining quality. The typical target thickness of formalin-fixed paraffin-embedded kidney sections is 2 μm. Slides are placed in a heated silver solution and microwaved to achieve a stirring effect and shorten the reaction time. The silver reaction can be easily controlled at 45°C, although the reaction temperature typically reaches 65°C. The application of thiosemicarbazide after PAM staining is extremely useful in microscopy. At high temperatures, the methenamine silver reaction clearly shows the mesangium, glomerular basement membrane (GBM), and renal tubular brush border (RTBB) because of their different color tones. Optimal staining resulted in black staining of the GBM, but brown staining of the RTBB. In conclusion, optimal visualization with reproducible results is obtained by using thiosemicarbazide at high temperatures after PAM staining compared with conventional silver staining.</p>