Polyclonal rabbit anti human cd3

For hematoxylin and eosin, Ki67, and CD3⁺ staining, paraffin-embedded fixed skin was cut to 2-μm thickness. Heat-induced antigen retrieval was performed in pH 6 sodium citrate buffer for 100 seconds at 125 °C under pressure. Polyclonal rabbit anti-human CD3 (1:100 dilution) and polyclonal rabbit anti-Ki67 (1:400) (both Dako, Carpinteria, CA), or negative controls, were used and counterstained with hematoxylin.

The serial slices used for histopathological analysis were immunostained with polyclonal rabbit anti-human CD3 (Dako, 1:100), polyclonal rabbit anti-human CD20 (Thermo Fisher, 1:100), monoclonal mouse anti-human MHCII (Dako, 1:40), monoclonal mouse anti-human Ki67 (Dako, 1:10), and monoclonal rabbit anti-human vWF (Dako; 1:3200) antibodies. Immunolabeling was achieved with a high-sensitive horseradish spell out (PO) mouse or rabbit diaminobenzidine kit, with blocking of endogenous PO (Envision DAB+kit; Dako) in an autoimmunostainer (Cytomation S/N S38–7410-01; Dako). An antibody diluent (Dako), with background-reducing components was used to block hydrophobic interactions. The average of three fields from each slice was used to quantitatively evaluate different immunohistological parameters and all measurements were performed with a computer-based program (Leica microscope DM LB2 with Leica Application Suite LAS V4.0) using 20× magnification.

Pancreases were fixed in neutral-buffered 10% formalin solution (Sigma), embedded in paraffin blocks, and cut in 5-μM sections. H/E staining was performed using standard protocols. For immunohistochemistry, sodium citrate buffer was used for antigen retrieval. The following antibodies were used: polyclonal rabbit anti-human CD3 (Dako, 1/200), rabbit anti-Ki67 (Abcam, 1/100), and biotinylated goat anti-rabbit (Abcam, 1/200). Biotinylated antibody was detected with the ABC system using diaminobenzidine as substrate (Vector Laboratories). Images were acquired with a Leica DM2500 microscope fitted with a 20× magnification lens. Reactive cells from 10 microscope fields per pancreas were counted using ImageJ. Results are shown as the number of reactive cells per mm² of tissue.

After the three-time vaccination of PBS, RFP, hFTN, or hFTN-RFP, the LN tissues were fixed in neutral buffered formalin and embedded in paraffin for immunofluorescence double-staining. The tissue slides (5 μm-thick) were deparaffinized and rehydrated, and the antigens were retrieved in boiling Tris-EDTA buffer (pH 9.0) for 10 min. The slides were first incubated in monoclonal mouse anti-human CD79α (1:200 diluted in PBS; DakoCytomation, Carpinteria, CA) for 2 h, and the fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG2b (1:300 diluted in PBS; Santa Cruz biotechnology, Santa Cruz, CA) was treated for 40 min for B cell detection. Then, polyclonal rabbit anti-human CD3 (1:200 diluted in PBS; DakoCytomation) and CFL555-labeled mouse anti-rabbit IgG (1:300 diluted in PBS; Santa Cruz biotechnology) was applied in consecutive order for T cell detection. The slides were covered with permount mounting medium containing DAPI for nuclear stain, and the fluorescence images were acquired using IX81-ZDC focus drift compensation microscope and digital image transfer software (Olympus). Eight random fields (×200 magnification) were selected in each slide, and the B and T cells were counted using imaging analysis software (Image Pro Plus 4.1; Media Cybernetics, Silver Spring, MD).

FFPE samples from 31 cats with T-cell lymphomas, 29 cats with B-cell lymphomas (Table 2), and 11 cats with reactive lymphoid proliferations (Table 3) were included in the study. The diagnosis of lymphoma and reactive lymphoid lesions was made by histology [according to WHO criteria (2 )] and immunohistochemistry against the T-cell marker CD3 (polyclonal rabbit anti-human CD3, Dako, Hamburg, Germany) and the marker CD45R (rat anti-mouse CD45R, clone B220/RA3-6B2 [Ly 5], Cedarlane, Burlington, Ontario, Canada) to identify B cells (28–31 (link)) according to routine protocols (32 (link)). All cases were reviewed by two senior pathologists (KK, MH), and only cases with an unequivocal diagnosis were included.