Saturated aqueous picric acid solution

Tissues were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 24 h, and then processed for embedding in paraffin. Tissue sections (5 μm) were cut and deparaffinized in xylene, followed by serial (100%, 95%, 70%, and 50%) alcohol washes to rehydrate, and were stained with hematoxylin and eosin (H&E). The rehydrated sections were boiled in antigen unmasking solution (Table 1) (Vector Laboratories) in a pressure cooker for 15 min. After blocking with 5% goat serum in PBS for 20 min, primary antibodies in 5% goat serum and 0.1% Triton X-100 in PBS at the indicated dilutions (Table 1) were applied to the sections and were incubated overnight at 4°C. The detection was performed using VECTASTAIN Elite ABC HRP kit (Vector Laboratories) according to manufacturer's instructions and the sections were counterstained with hematoxylin. Tunel assays were performed as described previously (Gavrieli et al. 1992 (link)). For Sirius red staining, following deparaffinization, the sections were stained with Picro-Sirius red solution containing 0.1% Direct Red 80 (#365548, Sigma) and 0.1% fast green (F7252, Sigma) in 1.2% saturated aqueous picric acid solution (Sigma 197378) for 1 h. Sections were rinsed with water, dehydrated, and mounted with Vecta Mount (Vector Laboratories).