Rat anti mouse cd3

Paraffin-embedded murine tissues were cut at 3 um and placed on super-frost slides. After dewaxing and rehydration, antigen unmasking was performed with Decloaking Chamber in DIVA Buffer 1X (DV2005L2J Biocare Medical, Pacheco, CA, USA) (3 min at 125 °C, 5 min at 90 °C) (CD31, CD3, Ly6G); for IBA1 staining, antigen unmasking was not performed. Endogenous peroxidases were blocked with 2% H₂O₂ for 20 min and then rodent block M (for IBA1) or PBS/BSA (bovin serum albumin) 2% (for CD31, CD3, and Ly6G staining) were used to block unspecific binding sites. Sections were incubated with the following antibodies: rabbit anti-mouse IBA-1 (1:250, Wako), goat anti-mouse CD31 (1:1000, R&D), rat anti-mouse CD3 (1:1000, Serotec), and rat anti-mouse Ly6G (1:200, BD Biosciences). All the primary antibodies were incubated for 1 h in a humid chamber at room temperature. As secondary antibody, we used a Rat on Mouse HRP polymer kit (Biocare Medical) (CD3, Ly6G), Goat on Rodent (Biocare Medical) (CD31), and Mach1 (Biocare Medical) (IBA1). Reactions were developed with 3,3′-diaminobenzidine, DAB (Biocare Medical) and then counterstained with hematoxylin and mounted with Eukitt.

Spleens were processed and stained as described previously (32 (link)). In short, spleens were frozen in liquid nitrogen and 5.5-μm-thick sections were cut and allowed to dry before fixation in acetone for 20 min at 4°C. In all cases, antibodies were added for 45 min of incubation at room temperature. Sheep anti-mouse IgD (Abcam) was detected with peroxidase-labeled donkey anti-sheep antibody (Jackson ImmunoResearch, Inc.) and developed with 3,3′-diaminobenzidine tablets (Sigma). CD3 and peanut agglutinin (PNA) were detected with rat anti-mouse CD3 (AbD Serotec), biotinylated rabbit anti-rat (Dako), and biotinylated rabbit anti-PNA (Vector Laboratories) antibodies. Biotinylated Ab binding signal was developed by using a streptavidin-biotinylated AP complex (Vectastain; Vector Laboratories) with Naphthol AS-MX phosphate, levamisole, and Fast Blue Salt.

Animals were perfused transcardially through the ventricular catheter with ringer (containing heparin). Spinal cord tissue was isolated, snap-frozen, and sectioned with a Leica CM1900UV cryostat (Leica Microsystems, Wetzlar, Germany) to obtain 8 µm slices. Dried cryosections were fixed in acetone for 10 min and subsequently blocked for 0.5 h with 10% normal serum. Cryosections were incubated overnight at 4°C with primary antibodies. Next, secondary antibodies were added for 1 h. Primary antibodies used were rat anti-mouse CD3 (1:150; AbD Serotec) and rat anti-mouse F4/80 (1:100; AbD Serotec). Goat anti-rat Alexa Fluor^®555 (1:400; Invitrogen) was used as a secondary antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; 1:2,500; Sigma-Aldrich). Stained sections were visualized under a Nikon Eclipse 80i fluorescence microscope (Nikon, Kingston, UK). Sections were analyzed using NIS Elements viewer software 4.0 (Nikon).