Timm Staining for Mossy Fibers

Timm staining is based on the staining of Zn²⁺-containing mossy fibers by a sulfide/silver stain as described previously. Briefly, the mice were deeply anesthetized and then fixed by transcardial perfusion with 0.9% NaCl followed by 0.3% Na₂S in 100 mM phosphate buffer (PB) and 4% paraformaldehyde in 100 mM phosphate buffer (PB). After perfusion, the brains were postfixed in 4% paraformaldehyde overnight at 4°C. Coronal sections were cut at 30µm on cryostat and stained for mossy fibers using Timm staining. The processing solutions were obtained from Sinopharm Chemical Reagent Co., Ltd., China and were as follows: 50% Arabic gumc, 15ml Citric acid buffer (3.825g citric acid,3.525g sodium citrate),45ml 5.67% hydroquinone, and 0.5ml 17% silver nitrate (51 (link)–53 (link)).Then stained for 40–60 min at 37°C. After rinsing, the sections were dehydrated in alcohol, cleared in xylene, and mounted on slides with permount. Timm staining was analyzed at a magnification of 40 and 100 using an OLYMPUS PM20 automatic microscope (Olympus, Japan).