Rabbit anti mouse cd3

Polymorphonuclear neutrophils (PMNs) were stained with rat anti-mouse antibodies to granulocyte marker Ly-6C/6G (Gr-1). Macrophages were stained with rat anti-mouse Mac-3 (BD Biosciences Pharmingen), CD3-positive cells with rabbit anti-mouse CD3 (Abcam), and GFP-positive cells with chicken anti-GFP (green fluorescent protein) antibodies (Aves Labs), respectively. A standard ABC vector kit (Vector Laboratories, Burlingame, CA) and a DAB reagent were used. To visualize CD3-positive cells a TSA Biotin System (Perkin Elmer) was used to enhance staining. Specificity of the staining was confirmed by using isotype control antibody. PMNs were counted manually in the entire section (outside and inside of the tracheal lumen) of the allograft, CD3-positive cells, and macrophages were counted in 4 random high-power fields under a microscope.

All tissues used for immunohistochemistry for histopathology were immersion fixed in 4 % paraformaldehyde. Tissue used for sections was embedded in OCT media. 7 μm sections were obtained and either stained with hematoxylin and eosin or processed for immunohistochemistry. Primary antibodies used include rabbit anti-Iba1 (Wako Chem, Osaka, Japan), CD3 (Rabbit anti-mouse CD3, Abcam, Cambridge, MA) and CD19 (rat anti-mouse CD19, Abcam). Secondary antibodies used were either goat anti-rabbit Cy3 or goat anti-rat Alexa 488 diluted 1:300 (both life technologies). It was also necessary to enhance the endogenous signal of DsRed⁺ lymphocytes through immunohistochemical approaches. Sections were incubated with the primary antibody mouse-anti DsRed (St. Cruz Biotechnologies, Dallas, TX) or rabbit-anti red fluorescent protein (Abcam) and counterstained with DAPI (Sigma, St. Louis, MO) to facilitate orientation.
Retinal whole mounts were preserved in 4 % paraformaldehyde. Retinas where preincubated in 0.3 % Triton X-100/PBS (Sigma) for 4 h and blocked in 0.1 % BSA/0.3 % Triton X-100/PBS for 1 h. Primary antibodies were diluted 1:300 in 0.3 % Triton X-100/PBS and incubated for 18 h at 4 °C. After extensive washing in PBS retinas were incubated with the secondary antibodies (1:200 in PBS) for three hours. Retinal wholemounts were coverslipped and images were taken on an Olympus BX41 microscope.