Mayer s hematoxylin stain

The intestinal tissue was removed and cut into sections in 5 mm thickness, and immediately fixed in 4% paraformaldehyde in PBS. 5 μm sections were cut and stained with hematoxylin and eosin (HE) for histological examination. For immunohistochemistry, sections were stained using the peroxidase-labeled, peroxidase, anti-peroxidase (PAP) antibody method (Dako REAL peroxidase blocking solution S2023, Glostrup, Denmark) with an anti-PCNA antibody (1:100, Santa Cruz Biotechnology, INC, sc-56), anti-HSP70 (1:100, Cell Signaling, #4872), and anti-HSP90 (1:100, Santa Cruz Biotechnology, INC, sc-7947). Mayer's hematoxylin stain was used for nuclei staining (Muto Pure Chemicals Co., Tokyo, Japan). For immunofluorescence, Anti-caspase-3 (1:100, Cell Signaling, #9664) was purchased. Stained slides were assessed using BZ-9000 fluorescence microscope (Keyence, Osaka, Japan).