Rabbit anti human cd3

Paraformaldehyde-fixed sections were paraffin embedded, sectioned and stained with haematoxylin and eosin for enumeration of eosinophils and villus: crypt ratios. An additional section from the jejunum was processed for immunohistochemical determination of T-cells, macrophages and Foxp3⁺ cells using the follow antibodies, respectively: rabbit anti-human CD3 (Dako), mouse anti-human macrophage (clone MAC387, Abcam) and mouse anti-human Foxp3 (clone 22510, Abcam). Appropriate isotype controls were used to determine antibody specificity. Bound antibodies were visualised with the Ultravision LP Detection System and aminoethyl carbazole substrate (ThermoFisher Scientific, Denmark). The Carnoys-fixed jejunum section was stained with Toluidine blue for mast cell enumeration. For determination of cell numbers, 10 random fields of each tissue section were counted by a single blinded observer using a calibrated counting grid covering a total area of 0.25mm² at 400x magnification. Cell counts were expressed as cells/mm² tissue. For villous: crypt ratios, 10 well-orientated villus: crypt units were randomly selected from each tissue section by a blinded observer. The sections were photographed using a Leica DFC480 camera and measurements performed using LAS v4.6 software (Leica, Switzerland).

Tissue sections of the paraffin-embedded biopsies were stained with haematoxylin–eosin (HE) for the detection of eosinophils, toluidine blue staining for mast cells and two immunohistochemical stainings (CD3 staining for T-cells and CD20 staining for B-cells), based on Dreesen et al. [31 (link)]. In short, skin tissue sections of 4 μm were mounted on APES-coated slides, blocked with H₂O₂ and stained with polyclonal rabbit anti-human CD3 (Dako, Belgium) or rabbit anti-human CD20 (Sigma-Aldrich, USA) antibodies. T- and B-cells were visualized by adding peroxidase labelled goat anti-rabbit antibodies (Dako, Belgium), diaminobenzidine tetrahydrochloride (DAB; Dako, Belgium) and by performing a counterstaining with haematoxylin. Eosinophils, mast cells, T-cells and B-cells were quantified by taking two random pictures per tissue slide at 400× magnification on a LEICA light microscope and counting the positive cells on two tissue slides per animal. Results were expressed as the number of cells per 10⁵ μm² tissue surface.

Tissues were collected and placed in neutral-buffered formalin for paraffin embedding. Sections were cut at 5 µm, deparaffinized and stained with hematoxylin and eosin, or blocked with 5% normal goat serum and 5% bovine serum albumin for immunostaining with primary antibodies specific for YFV antigen (mouse anti-YF clone 3A8.B6; 1.5 µg/µL, a generous gift from Dr. Ian Amanna), B cells (mouse anti-human CD20, Dako; 1∶475), or T cells (rabbit anti-human CD3, Dako; 1∶200). Secondary antibodies used were: biotinylated goat-anti-mouse IgG and biotinylated goat-anti-rabbit IgG (Vector; 1∶300). DAB chromagen with hematoxylin counterstain (Vector) was used to visualize CD20+ B cells and CD3+ T cells. VIP substrate with methyl green counterstain (Vector) was used to visualize YFV antigen. The sections were then analyzed and images captured using an Axioplan microscope (Carl Zeiss) with a Spot Insight camera (Diagnostic Insturments Inc.)

Archived formalin fixed and paraffin embedded (FFPE) tissues were identified by searching the Veterinary Medical & Administrative Computer System at UCD School of Veterinary Medicine. Five μm tissue sections were cut and mounted onto glass slides. Tissues were deparaffinized with xylene and a serial ethanol dilution. Slides were boiled at 95°C for 20 minutes (Target Retrieval Solution, DAKO, Carpinteria, CA) following blocking with 15% donkey serum (Jackson ImmunoResearch Laboratories Inc, West Grove, PA) and 1% Fc blocker (Miltenyi Biotec) for 45 minutes. Slides were than incubated with rabbit anti-human CD3 (DAKO) and goat anti-human IL17 (R&D) antibodies at 4°C overnight. Slides were then washed and stained with donkey anti-rabbit AF488 (Invitrogen) and donkey anti-goat AF555 (Invitrogen) for 1 hour at room temperature in the dark. Finally, slides were mounted with DAPI containing mounting media (Vector Laboratories, Burlingame, CA). Images were acquired with a LSM 700 confocal microscope (Zeiss, Pleasanton, CA).

Detailed methods can be found in supplemental methods. Antibodies include: rat anti-mouse CD31, 1:50 (BD Pharmingen); mouse anti-mouse alpha smooth muscle actin, 1:100, (Abcam); rabbit anti-mouse γH2AX, 1:750 (Millipore); rabbit anti-mouse Ki67, 1:250 (Novus); rabbit anti-human CD3, 1:200 (Dako).
For quantification, 5 random 10× magnification pictures were taken of each slide and the area of CD31⁺ structures, number of visible lumens, vessels, and vessels > 100 μm was counted. Values for each of the 5 sections were averaged to obtain one value for each tumor. Individual averages for all tumors within a treatment group were then averaged to determine the group average and SEM.

Serial tissue sections (4 µm) were deparaffinised in xylene, dehydrated in 100% ethanol and endogenous peroxidase was blocked in methanol with 0.3% H₂O₂ for 30 min. Antigen retrieval was performed by heat inactivation in 10 mM Tris-EDTA buffer (pH 9.0; boiled for 10 min) for immunostaining of CD3. No antigen retrieval was required for CD45. Subsequently, tissue sections were incubated with either rabbit anti-human CD3 (1:50 dilution; Dako Agilent, Amstelveen, The Netherlands) or mouse anti-human CD45 (1:50 dilution; Dako Agilent) for 60 min at room temperature. After a wash in phosphate-buffered saline the slides were incubated with Envision HRP anti-mouse/anti-rabbit (undiluted, Dako) for 30 min. The slides were counterstained with haematoxylin, dehydrated and covered. For fibrosis analysis, tissue cross-sections were stained using the histological Elastica von Gieson (EvG) staining method, according to the standard protocol. Negative controls were included with each staining and showed no staining (not shown). All slides were scored by two independent observers (L. Wu and P.A.J. Krijnen; inter-observer variation <10%).