Initial screening for insulin, MHC class I, and CD8. For characterization of the sections before tetramer staining, staining for insulin, CD8, and HLA-ABC (all at room temperature) was performed using the antibodies listed in Table 3. Fixation was with acetone and sections were blocked with goat serum in a standard immunofluorescent staining protocol. An islet was determined as hyperexpressing MHC class I based on a threshold of three unequivocally positive cells, which was the maximum number found in all control islets examined.Table 3.Antibodies used for characterization of the sections prior to tetramer stainingAntigenPrimary antibodyDetection antibodyInsulinPolyclonal guinea pig anti-insulin (Dako; 1/140 dilution, 1-h incubation)Polyclonal goat anti–guinea pig IgG, highly cross-adsorbed, Alexa Fluor 488 (Invitrogen; 1/1000 dilution, 30-min incubation)HLA-ABCMouse monoclonal (clone W6/32) IgG2a against a monomorphic epitope on the 45 kD polypeptide products of the HLA-A, B and C loci (Dako; 1/100 dilution, 1 h incubation)Polyclonal goat anti–mouse IgG2a, isotype-specific, Alexa Fluor 594 (Invitrogen; 1/1000 dilution, 30-min incubation)CD8Mouse monoclonal (clone HIT8a) IgG1 against the CD8 alpha subunit (BD; 1/100 dilution, 1-h incubation)F(ab’)2 fragment of goat anti-mouse IgG, Alexa Fluor 594 (Invitrogen; 1/1000 dilution, 30-min incubation)