Ihc antibodies

We purchased angiotensin II (A9525) and elastase (E7885) from Sigma-Aldrich (St. Louis, Missouri, USA) and Alzet osmotic pumps (200 µL, 0.5 µL/h) from Durect corporation (Cupertino, California, USA). The 10 µL model 701 syringe with a 26-G 2.0-inch point-style-3 Hamilton replacement needle was supplied by Fisher Scientific (Hampton, New Hampshire, USA). We purchased IHC antibodies from Abcam (Cambridge, UK) and Cell Signaling Technology (Danvers, Massachusetts, USA).

The −80°C-stored samples were homogenized in radio-immunoprecipitation assay (RIPA) buffer with 10 mM phenylmethylsulfonyl fluoride (PMSF). Equal amounts of protein lysate (50 μg) were separated by 10% (w/v) sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (Sangon Biotech, Shanghai, China) and electro-transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA, United States). Membranes were blocked with 3% bovine serum albumin (BSA) (BBI, Shanghai, China) for 2 h at 25°C and incubated with primary antibodies (diluted at 1:500 in PBS) of T1R2, T1R3, GNAT3 (same as IHC antibodies, Abcam, Cambridge, United Kingdom), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (lot: HG0718, HuaBio, Hangzhou, China) for 15 h at 4°C. The incubated membranes were washed with Tris-buffered saline with Tween (TBST) buffer and second-incubated with NIR-secondary antibody Odyssey IRDye 680RD goat anti-rabbit IgG (lot: C51104-08, LI-COR, Lincoln, NE, United States) for 1.5 h at 25°C, respectively. The bands were washed in TBST buffer (five times) and visualized with an Odyssey CLx imaging system (LI-COR, Lincoln, NE, United States), and the intensities of blots were calculated with target protein bands to corresponding GAPDH.

IHC was performed as described previously (Mori and Cardiff 2016 (link)). Tissues are loaded into embedding cassettes. Then the paraffin sections were deparaffinized and rehydrated. The antigen retrieval was performed by immersing the slides in sodium citrate buffer (10 mM, pH = 6.0) and heating in a microwave oven for 10 min. After cooling, the slides were washed three times with phosphate-buffered saline (PBS) for 5 min. Endogenous peroxidase was blocked using 3% hydrogen peroxide at room temperature for 10 min, followed by washing with PBS. Then the slides were blocked with normal goat serum at room temperature for 20 min. Primary antibodies were added to each section and incubated at 4°C overnight, followed by incubation with secondary antibody at 37°C for 30 min. The tissue slide was then immersed in the DAB solution and rinsed with distilled water. IHC antibodies were obtained from Abcam. Image data were analyzed using Indica Labs (United States). All statistical analyses were performed using SPSS software 23.0 (IBM Corporation, United States). The differences in immunopositivity and staining were assessed using Student’s t-test. All IHC tests were carried out blind.