Rabbit anti cd3

Heart allografts were fixed with 4% paraformaldehyde and their paraffin-embedded sections were stained using H & E (hematoxylin and eosin) stain. Tissue injury by rejection was semiquantitatively assessed according to a previous study [24 (link)]. Immunohistochemical staining was also performed using rabbit anti-CD3 (DAKO, Glostrup, Denmark) or anti-mouse F4/80 (clone BM8, Thermo Fisher Scientific), followed by a Dako Real Envision HRP kit (DAKO). The stained slides were assessed using an Olympus BX40 light microscope (Olympus, Japan), and images were analyzed with Image-Pro 5.1 software (Media Cybernetics, Rockville, MD, USA). All histological analyses were performed by two independent researchers blinded to the treatment group.

Tumors were fixated by perfusion using ZN²⁺ fixative. After fixation and paraffin embedding, sections were deparaffinized using xylene and graded alcohol before hydration and washing with PBS. Antigen retrieval was performed in a sodium citrate buffer (pH 6) in a microwave oven, and endogenous peroxidase was subsequently blocked by 0.3% H₂O_2. Sections were incubated overnight at 4°C with a primary antibody (rabbit anti-CD3; Dako). An anti-rabbit-horseradish peroxidase SuperPicTure Polymer detection kit (Invitrogen, San Francisco, CA, USA) was used as a secondary antibody. A matched isotype control was used as a control for non-specific background staining, and a routine standard staining was performed with hematoxylin and eosin.

Spleen tissue sections were fixed with 4% paraformaldehyde in PBS and embedded in paraffin or by 1:1 acetone/methanol fixation for frozen sections. Paraffin sections were stained with hematoxylin and eosin. Histological examination of tissue sections was done in a blind manner. Frozen sections were stained with TRITC-conjugated peanut agglutinin (PNA; Sigma), Alexa Fluor 647-labeled anti-IgD (Biolegend), and FITC-labeled anti-CD4 mAb (BD Biosciences). Paraffin sections were stained using rat anti-mouse B220 (BD Biosciences), which was detected using anti-rat FITC-labeled IgG (Sigma), and rabbit anti-CD3 (DAKO), which was detected using anti-rabbit Alexa Fluor 647-labeled IgG (Invitrogen), together with TRITC-conjugated PNA (Sigma). Stained sections were washed in phosphate-buffered saline (PBS) and mounted using Vectashield (Vector Laboratories, Burlingame, CA).

The endogenous biotin-blocking kit and the streptavidin conjugated with Texas Red were purchased from Molecular Probes. Biotinylated goat anti-mouse IgG and VECTASHIELD Mounting Medium with Dapi were obtained from Vector Laboratories. Rabbit anti-CD3 was purchased from Dako. Alexa Fluor 488 goat anti-rabbit IgG was obtained from Life Technologies. Dr. Bettina Wagner (Cornell University) supplied the murine anti-equine monoclonal antibodies (supernatants from clones IL-17A76, 79–3 and 80–1). The monoclonal antibodies were made as part of the US-wide initiative, “The U.S. Veterinary Immune Reagent Network.” The specificity of the equine IL-17A antibodies had been previously tested using various equine recombinant equine cytokines by ELISA. Recombinant cytokines were IgG or IL-4 fusion proteins and included IL-4/IL17, IL-4/IFN-γ, IL-4/IL-5, IL-4/IgG1, IL-2/IgG1 and IL-10/IgG4. The anti-IL-17A antibodies detected the IL-4/IL-17 protein but none of the other IL-4 or IgG fusion proteins. In addition, the IL-17A antibodies were validated in non-stimulated and stimulated equine PBMC by intracellular staining and flow cytometric analysis as previously described [11 (link)]. Hybridoma medium to grow the antibodies was purchased from Life Technologies.

The number and type of inflammatory cells were characterized in the prostate of Akita and WT mice. Three random areas from each prostatic lobe from each animal were acquired (WT: n = 4; diabetic: n = 4). Neutrophils were identified and quantitated in H&E stained sections. Immunohistochemistry was used to quantitate monocytes/macrophages, T lymphocytes, B lymphocytes, and fibrocytes. Briefly, sections were blocked for 4 hours in PBS containing 10% donkey serum and 1% BSA (both from Sigma-Aldrich, St. Louis, Missouri) was followed by primary antibodies-rat anti-F4/80 (1 : 50, eBioscience, San Diego, CA), rabbit anti-CD3 (1 : 100, Dako, Carpinteria, CA) and goat anti-CD20 (1 : 100, Santa Cruz, Santa Cruz, CA), and rat anti-CD45 (1 : 100, Abcam, Cambridge, MA) and rabbit anti-vimentin (1 : 100, Abcam, Cambridge, MA) overnight at 4 degrees. Secondary antibodies-donkey anti-rat Alexa 594, donkey anti-rabbit Alexa 594, donkey anti-rat Alexa 488, and donkey anti-goat Alexa 488 (1 : 100, Invitrogen, Grand Island, NY) were incubated for one hour at room temperature. Four μg/mL of Hoechst 33258 (Sigma-Aldrich, St. Louis, Missouri) was incubated for 10 minutes. For quantification of each cell type, three random areas from each prostatic lobe from each animal were acquired using Nikon eclipse Ti-U microscope.