Histostain plus kit

Routine serial sections of formaldehyde-fixed, paraffin-embedded tissue blocks of the follicles were cut into 4-μm-thick slices and then deparaffinized with xylene and rehydrated through a graded ethanol series. After pressure cooking in 10 mM sodium citrate buffer, the sections were incubated in 3% hydrogen peroxide for 15 min to block endogenous peroxidase activity, and incubated in 10% goat serum for 15 min prior to incubation with the rabbit anti-chicken PTHLH or PTH1R polyclonal IgG antibody (1:100) for 2.5 h at 37°C. The sections were then incubated with the secondary antibody for 30 min and the avidin-biotin-peroxidase complex for another 30 min according to the Histostain-plus kit instructions (Zhongshan Golden Bridge Biotechnology, Beijing, China). Finally, the immunoprecipitates were visualized using a diaminobenzidine (DAB) kit (Zhongshan Golden Bridge Biotechnology, Beijing, China). The slides were counter stained with hematoxylin after immunostaining, dehydration and covering. Negative controls were prepared in each case by replacing the primary antibody with PBS; no specific staining was found in the control slides.

Metformin, compound C, and human recombinant insulin were from Sigma‐Aldrich (St. Louis, MO, USA). Anti‐pan‐FOXO1 (N18), anti‐ki‐67, and Anti‐p‐AMPK (Thr172) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) for IHC. Anti‐p‐AMPK, anti‐pan‐AMPK (Thr172), anti‐p‐FOXO1 (Ser256), anti‐p‐AKT (Ser473), and anti‐pan‐AKT antibodies were from Cell Signaling Technology (Beverly, MA, USA). Anti‐pan‐FOXO1 (H128) antibody for Western blotting and immunofluorescence staining was from Santa Cruz Biotechnology. The BCA Protein Assay Kit for quantification of total protein and the Enhanced Chemiluminescence Detection Kit were from Pierce (Rockford, IL, USA). The Histostain‐Plus Kit was from Zhongshan Golden Bridge Biotechnology (Beijing, China). The annexin V–FITC/propidium iodide Apoptosis Detection Kit was from BD Biosciences (San Jose, CA, USA). The Lipofectamine 2000 reagent were from Invitrogen (Burlington, ON, Canada). The non‐specific scramble siRNA control and FOXO1‐specific siRNA were purchased from Cell Signaling Technology. All other chemicals were purchased from Sigma‐Aldrich.