Hsp70

The small intestinal tissue fixed in Bouin’s solution were trimmed, dehydrated by gradient alcohol, transparented by xylene, and then embedded in paraffin. The wax blocks were continuously sliced with a thickness of 5 μm. Six consecutive slices treated with polylysine were selected for Hsp70 immunohistochemical staining. In order to obtain the antigen, the tissue sections were pretreated with sodium citrate buffer. The buffer concentration was 0.01 mol/L, pH was 6.0, and the treatment time was 20 min. In order to block the nonspecific binding, 10% normal goat serum (zsgb-bio, Beijing, China) was used to culture. In order to block the endogenous peroxidase activity, 10% hydrogen peroxide (H₂O₂) was used for 1.5 h. Then, the polyclonal antibody Hsp70 (1:50, bm0368, BIOSS, Beijing, China) was incubated with sections at 4 °C for 12 h. The samples were tested for immunohistochemical analysis using tissue staining SP Kit (spn-9001, zsgb-bio, Beijing, China); polink-2 plus Polymer horseradish peroxidase (HRP) rabbit or mouse primary antibody detection system (pv-9002, zsgb-bio, Beijing, China). The sections were soaked in diaminobenzidine tetrachloride, and the DAB Kit (pa110, Tiangen, Beijing, China) was used, and the time was no more than 3 min. The distribution of positive cells should be dehydrated and sealed, and observed by microscope.

Total protein was extracted from cells using cell lysis buffer (Boster) containing protease inhibitors (Boster, Wuhan, China). Total proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis on 10% gels, and the separated proteins were transferred to polyvinylidene fluoride membranes (Millipore, NY, USA). After blocking with 3% BSA for 2 h, the membranes were incubated with primary antibodies against the following proteins: YAP (1 : 500, Boster, Wuhan, China), COL I (1 : 1000; Santa Cruz, USA), osteocalcin (OCN; 1 : 1000; Abcam, UK), RUNX-2 (1 : 1000; Abcam, UK), osteopontin (OPN; 1 : 1000; Boster), TG2 (1 : 1000; Santa Cruz, USA), HSP70 (1 : 500, Bioss, Beijing, China), and YAP (1 : 500, Boster, Wuhan, China). HRP-conjugated goat anti-rabbit IgG (1 : 5000; Boster) was applied as a secondary antibody and incubated with the membrane for 1 h at room temperature. Immunoreactive bands were detected using enhanced chemiluminescence reagents (Millipore, NY, USA). Signal intensity was measured using a Bio-Rad XRS chemiluminescence detection system (Bio-Rad, CA, USA). Actin or β-tubulin served as the loading control.