Alexafluor647 conjugated anti cd3a

Spleens, pancreata and hearts were harvested and froZEN directly in OCT, 8 μm sections were taken and were fixed for 5 min with cold acetone prior to staining. Brains were fixed in 4% paraformaldehyde (Sigma-Aldrich), equilibrated in 20% sucrose (Sigma-Aldrich) and embedded in OCT prior to sectioning. 10 μm sagittal brain sections were incubated with 2% goat serum, 1% BSA, Fc-Block 1:1000, 0.05% Tween-20 and 0.1% Triton X-100 in PBS before staining and stained in 1% goat serum, 1% BSA, 0.5% Triton X-100, Fc-Block 1:1000. RFP staining was performed with mouse RFP (GT1610) antibody (Abcam) and a secondary goat anti-mouse AF-568 (Life Technologies). Vessels in the brain were stained with rabbit anti-collagen IV, polyclonal (BioRad, #2150-1470) and a secondary goat anti-rabbit AF-647 (Life Technologies). T-cell staining was performed for 2 hours with either AlexaFluor594-conjugated anti-CD8a (clone 53-6.7 BioLegend) or CD3e staining with AlexaFluor647-conjugated anti-CD3a (clone 17A2 BioLegend) and DAPI (Vector Laboratories) for nuclei labeling. Sections were blocked with 2% rat serum and 0.5% BSA in PBS before staining. Images were obtained with an upright wide-field microscope (Nikon) equipped with a 20x lens, and analyzed with NisElements software, or with an Zeiss LSM 780 confocal microscope and analyzed with ZEN (Zeiss) and ImageJ softwares.