Alexa 488 conjugated donkey anti mouse

Laboratory Rodent Diet 5001 was purchased from EL Mel (St. Louis, MO) and Sucrose Dustless Precision Pellets were from Bio-Serve (Flemington, NJ). Insulin rabbit mAb was obtained from Cell Signaling (Danvers, MA). Stretozotocin and monoclonal mouse anti-glucagon antibody were obtained from Sigma (St. Louis, MO). The secondary antibodies, Alexa 647-conjugated donkey anti-rabbit antibodies and Alexa 488-conjugated donkey anti-mouse were obtained from Jackson Immuno Research Laboratories (West Grove, PA). Schiff reagent, periodic acid, total cholesterol, high density lipoprotein (HDL)-cholesterol, and triacylglyceride (TAG) liquid reagents and their respective standards were purchased from Fisher Scientific (Pittsburgh, PA) and alpha-amylase was from MilliporeSigma (Burlington, MA). Contour Next test strips were obtained from ADW Diabetes (Pompano Beach, FL). Rat hemoglobin A1c kit and control for the kit were obtained from Crystal Chem (Elk Grove Village, IL).

Sections were washed in PBS 3 × 10 min on a filter shaker. To accomplish the disruption of the cell membranes, the sections were treated in 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) detergent with PBS for 30 min on a filter shaker. To block non-specific binding sites, the sections were treated in 2% normal donkey serum (NDS) with PBS on shaker for 30 min. The slides were placed in 2% NDS in PBS containing primary antibodies: mouse anti-TH (Sigma-Aldrich, St. Louis, MO, USA, Cat. No.: T2928, in 1:1000 dilution) and rabbit anti-FOSB (Abcam, Cambridge, UK, Cat. No.: ab184938, in 1:6000 dilution) antibodies at RT overnight.
The following day, the sections were washed for 3 × 10 min in PBS and incubated for 3 h with the secondary antibodies (Alexa 488-conjugated donkey anti-mouse (Jackson Immunoresearch Europe Ltd, Ely, UK, Cat. No.: 715-545-150, in 1:500 dilution) and Cy3-conjugated donkey anti-rabbit (Jackson Immunoresearch Europe Ltd, Ely, UK, Cat. No.: 711-165-152, in 1:500 dilution) dissolved in PBS and 2% NDS. After 3 × 10 min washes in PBS, the sections were placed on gelatin-coated slides and dried for 1 h at RT in the dark, after which, they were covered with glycerol-PBS (1:1) and stored at −20 °C until the microscopic examination.