Anti insulin receptor

Four Epac1 floxed and Epac1 Cdh5 Cre-lox mice were euthanized with carbon dioxide and cervical dislocation. The eyes were removed and placed in 4% paraformaldehyde in PBS (1X; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.4) overnight. The next day, the eyes were transferred to 0.1 M PBS containing 30% sucrose for cryoprotection. Using a cryostat, ten-micron sections were cut and stored at 80°C until use. The sections were rinsed in PBS and put in 5% normal goat serum for 1 h at room temperature to block nonspecific staining, followed by incubation with isolectin GS-IB4 to label the retinal vasculature (Alexa Fluor 488 conjugate, 1 : 100, Life Technologies) [10 (link)] and anti-insulin receptor (1 : 500, Abcam) at 4°C overnight. After incubation, the sections were rinsed in 0.3% Triton/PBS followed by incubation with secondary antibody goat anti-rabbit conjugated to Alexa Fluor 594 (1 : 1000, Life Technologies) for 2 h at room temperature. Slides were rinsed in PBS and counterstained with 4′,6-diamidino-2-phenylindole (DAPI). The slides were examined with a Leica confocal microscope (Buffalo Grove, IL).

The proteins in the sample were harvested using RIPA lysis buffer (Merck Millipore, Burlington, MA, USA). The experimental procedure was also following our previous publication [24 (link)]. The antibodies used are listed as follows: anti-JAK1 (BD biosciences, East Rutherford, NJ, USA); anti-JAK2, anti-STAT1 (Cell Signaling, Beverly, MA, USA); anti-pSTAT1, anti-STAT3 (Cell Signaling, Beverly, MA, USA); anti-pSTAT3, anti-SOCS3 (Abcam, Cambridge, UK); anti-insulin receptor (Abcam, Cambridge, UK); anti-TLR-4 (Proteintech, Chicago, IL, USA); anti-IL-6 (Bioworld Technology, St Louis Park, MN, USA); and anti-β-actin (Santa Cruz, Santa Cruz, CA, USA) at room temperature (RT) for 1 h. After the incubation with the appropriate secondary horseradish peroxidase-conjugated IgG antibody (R&D Systems) for 30 min at RT, the protein bands on the membrane were detected with ECL-Plus Western Blot Detection system (GE Healthcare UK LTD) according to the instructions of the manufacturer. All experiments were replicated at least thrice.