Avidin biotin enzyme complexes

At the end of the study, pancreases (n = 3) were collected for histological assessment. Tissue was fixed in Z-fix, embedded in paraffin, and tissue sections were immunostained as described previously [20 (link)]. Primary antibodies were guinea pig anti-insulin (diluted 1:1000; Dako Agilent Pathology Solutions, Santa Clara, CA, USA) and mouse anti-glucagon (diluted 1:5000; Sigma). Appropriate biotinylated secondary antibodies and avidin–biotin–enzyme complexes were purchased from Vector Laboratories (Burlingame, CA, USA). Diaminobenzidine as the chromogen was purchased from BioGenex (Fermont, CA, USA). Tissue sections were counterstained with hematoxylin.

At the end of the experiment, pancreases (n = 4 per treatment group) were fixed in Z-fix and paraffin embedded for histological assessment. Tissue sections were immunostained for insulin and glucagon as described previously^{55 (link)}. Primary antibodies included guinea pig anti-insulin (diluted 1:1000; Dako Agilent Pathology Solutions, Santa Clara, CA) and mouse anti-glucagon (diluted 1:5000; Sigma). Biotinylated secondary antibodies and avidin–biotin–enzyme complexes were purchased from Vector Laboratories (Burlingame, CA). Diaminobenzidine was used as a chromogen (BioGenex, Fermont, CA). All sections were counterstained with hematoxylin. Apoptosis was determined using the DeadEnd^TM Fluorometric TUNEL assay (Apoptosis Detection System; Promega Corporation, Madison, WI) according to the manufacturer’s instructions. Negative controls included sections incubated without the TdT enzyme. For quantification, the total number of apoptotic cells per tissue section was counted and normalized to the total tissue area.