Guinea pig antibody to insulin

Antigen retrieval was performed on paraffin-embedded sections with Retrievagen A Solution (BD Biosciences), and endogenous biotin was blocked by Dual Endogenous Enzyme Blocking Reagent (Dako). Guinea pig antibody to insulin (Dako) or rabbit antibody to glucagon (Abcam) was added and detected with the EnVision Dual Link Kit (Dako) followed by staining with DAB Solution (Dako). Samples were counterstained with hematoxylin. A veterinary pathologist scored histopathological changes by blinded scoring of sections.

Antigen retrieval was mediated at 98°C for 45 min in formalin-fixed, paraffin-embedded sections. Sections were blocked with PBS containing 1% BSA and 5% normal goat serum, then incubated with the following primary antibodies overnight: guinea pig antibody to insulin (1:150; Dako); rabbit antibody to glucagon (1:50; Dako); mouse antibody to VP1, clone 5-D8/1 (1:50; Dako); rabbit antibody to cytokeratin 19 (CK19) (1:500; Abcam); and/or rabbit antibody to amylase (1:400; Abcam). Sections were incubated with the following secondary antibodies for 1 h at 1:1,000 dilution: Alexa Fluor-594 goat antibody to guinea pig IgG; Alexa Fluor-647 donkey antibody to rabbit IgG; and Alexa Fluor-488 goat antibody to mouse IgG (catalog #A11076, #A31573, and #A11029, respectively; Life Technologies). Sections were mounted with ProLong Gold Antifade Reagent with DAPI (Life Technologies). Immunofluorescence was imaged on a Leica SP8 confocal microscope and quantified using FIJI software (version 1.48p) using automatic thresholding followed by the measure area function (24 (link)). Wide-field images were acquired using a Nikon Eclipse Ni-U microscope with a ×4 plan objective using NIS-Elements imaging software (version 4.13). High-magnification wide-field images were acquired with a ×40 plan objective using QCapture Pro software (version 5.1).