Anti mouse cd3 clone 17a2

Following fixation with 10% buffered formalin, heart allografts were stained with hematoxylin and eosin (H&E), and Movat pentachrome. For immunoperoxidase staining, 4 μm formalin-fixed paraffin-embedded sections were dewaxed in five changes of xylene and brought down to water through graded alcohols. Heat-induced epitope retrieval was performed using a microwavable pressure cooker with tissue sections in Tris-EDTA buffer at pH 9.0. The following primary antibodies were used for staining: anti-rat/mouse Foxp3 (clone FJK-16s) (eBioscience, San Diego, CA, USA), anti-mouse CD3 (clone 17A2) (eBioscience), and anti-mouse B220 (clone RA3-6B2) (BD Biosciences). The detection system used was an HRP anti-rat IgG ImmPRESS kit (Vector Labs, Burlingame, CA, USA). Deposition of IgG in allografts was detected using an HRP anti-mouse IgG Impress kit (Vector Labs) without a primary antibody. After following the kit instructions, color development was performed with freshly prepared DAB (DAKO, Burlington, Canada). Morphometric analysis was performed at the STTARR facility (University Health Network, Toronto, Canada) following scanning with an Aperio ScanScope XT (Leica Biosystems, Wetzlar, Germany). Positively stained cells and IgG staining were quantified using TissueStudio (Definiens, Carlsbad, CA, USA).