Hrp sheep anti mouse

For nNOS detection, cells were lysed in 50 μl of RIPA buffer (150 mM NaCl,50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.4, 5 mM ethylene diamine tetra acetic acid, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM PMSF with a mix of protease inhibitors (Roche), and centrifuged 10 min 1500g at 4 °C.
Protein extracts (20 μg) were denaturated in Laemmli buffer 2× added of 10% of 2-mercaptoethanol 30 min at room temperature (RT) and incubated 15 min in ice and then 15 min at RT. Proteins were resolved by SDS–PAGE (4–12%, Invitrogen) and transferred to nitrocellulose. Membranes were blocked in tris-buffered saline 0.1% Tween-20 with 5% non-fat dry milk 1 h at RT and incubated overnight at 4 °C, with rabbit polyclonal nNOS antibody (R-20, Santa Cruz, 1:100) or with mouse monoclonal GAPDH antibody (MAB9748, Tebu-Bio, 1:8000). After being washed in TBS 0.1% Tween, membranes were incubated for 1 h at RT with secondary antibodies: goat anti-rabbit-horseradish peroxidase (HRP) (1/50000) or sheep anti-mouse HRP (1/15000) (Jackson Immunoresearch). Western blots were revealed with enhanced chemiluminescence (Thermo Scientific) with Image Quant LAS 4000 system (GE Healthcare Life Sciences).

To extract the proteins from the tissues, 3 IMN kidney tissues and 3 NATs were cut into pieces and lysed on ice using RIPA lysis buffer (Applygen, China) supplemented with Protease Inhibitor Cocktail, and then quantified using the BCA Protein Assay Kit. According to the expression of the internal reference protein GAPDH in the specimen, 90 µg extracted proteins from IMN kidney tissue and 10 µg extracted proteins NATs were separated by 12 % SDS-PAGE and transferred to a nitrocellulose (NC) membrane (Millipore, Darmstadt, Germany). After blocking with 5 % non-fat dry milk in tris-buffered saline and 0.1 % Tween 20 solution, membranes were incubated overnight at 4 ℃ with primary antibodies against the following proteins: MMP9 (ABGENT, China, 1:1000), CAPN1 (Abclonal, China, 1:1000), MMP9, MMP14, CTSS, and GAPDH (Proteintech, China, 1:5000). The membranes were then incubated with the appropriate HRP-Goat Anti-Rabbit (Elabscience, China, 1:2,500) and HRP-Sheep Anti-Mouse (Jackson ImmunoResearch, USA, 1:8,000) secondary antibody for 1 h at room temperature. The specific bands were detected using an ECL detection kit (Applygen, China) and captured on an ImageQuant LAS 4000 mini system (GE Healthcare, NJ, USA). Relative expression was determined by normalizing to GAPDH expression using the ImageJ software (Version 1.51j, National Institutes of Health, MD, USA).