H 10 sc 20175

Freshly isolated islets from 3–4 month old male mice were sonicated in 150–200 μl lysis buffer supplemented with protease inhibitor tablet (Roche Diagnostics, Basel, Switzerland). The cell extract was diluted by 1–1.5 volume Laemmli loading buffer containing 5% β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO) and boiled for 5 min before loading onto SDS-PAGE gels. Together with liver and visceral fat extracts, Western blotting was performed to quantify protein levels of 11β-HSD1 (1:250, H-10 sc-20175, Santa Cruz, Dallas, TX) and β-actin (Santa Cruz or Medimabs, Montreal, QC). Similarly, MIN6 cells were used to probe for total and cleaved caspase-3 (1:1000, Cell Signaling, Danvers, MA).

Paraffin sections of the pancreas taken from 3–5 month old, male MT-IGF and wild-type littermates were dewaxed, rehydrated, and blocked with 10% donkey serum, followed by incubation with rabbit anti-11β-HSD1 antibodies (1:100; H-10 sc-20175, Santa Cruz and ab83522, Abcam, Cambridge, MA) at 4°C overnight. After washing with PBS, sections were independently stained with anti-glucagon (C-18 sc-7779, Santa Cruz) and guinea pig polyclonal anti-insulin (ab7842, Abcam) followed by Alexa Fluor 594 conjugated donkey anti-rabbit IgG (H+L) and Alexa Fluor 488 goat anti-guinea pig IgG (Life technologies, Carlsbad, CA) [14 (link), 15 (link)]. The images were analyzed using Axioshop 2 plus microscope (Carl Zeiss, Jena, Germany), Retiga 1300 digital camera, and Northern Eclipse software (Empix Imaging, Mississauga, ON). Paraffin sections of liver and pancreata taken from 3–5 month old male wild-type mice were incubated overnight with anti-11β-HSD1 (ab83522) with or without a specific blocking peptide (ab99223) at 4°C. After washing, the sections were incubated with secondary antibody and stained with diaminobenzidine substrate (Vector Laboratories). The microscopic images were analyzed using BX61 UIS2 Optical System microscope (Olympus) and Olympus stream software.