Bx61 uis2 optical system microscope

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.

Formalin-fixed, paraffin-embedded tissues were microtome-sectioned to a thickness of 4 μm, placed on electromagnetically charged slides (Fisher Scientific; Waltham, MA, USA), and stained with hematoxylin & eosin for routine histologic analysis. Immunohistochemistry was performed using the Avidin–Biotin–Peroxidase complex system, according to the manufacturer's instructions (Vectastain Elite ABC Peroxidase Kit; Vector Laboratories, Burlingame, CA, USA). Following antigen retrieval, slides were washed with phosphate-buffered saline (PBS) and blocked in PBS/0.1% bovine serum albumin containing 5% normal goat serum for 2 h at room temperature, then incubated overnight with primary anti-KDM5A (RBP2) rabbit polyclonal antibody (1:500, Sigma-Aldrich; St Louis, MO, USA) or anti-chromogranin A mouse monoclonal (Dako, 1:500; Carpinteria, CA, USA). The following day, slides were incubated with biotinylated secondary antibodies, developed using a diaminobenzidine substrate, counterstained with hematoxylin, and mounted with Permount. Images were collected at × 200 and × 600 magnification using an Olympus BX61 (UIS2 optical system) microscope equipped with a high resolution Olympus DP72 camera and CellSense image capture software.