Ab828

Triple immunofluorescent staining was performed on 4-μm-thick FFPE sections. After antigen retrieval using Tris–ethylenediaminetetraacetic acid (EDTA) buffer (10 mM Tris plus 1 mM EDTA pH 9.0), rabbit anti-CD3 (ab828, Abcam, Cambride, UK), mouse IgG1 anti-FoxP3 (ab20034, Abcam) and goat anti-IL-17 (AF-317-NA, R&D Systems, Abingdon, UK) diluted in 1 % w/v bovine serum albumin (BSA) in phosphate-buffered saline (PBS) were incubated at room temperature overnight. Alexa Fluor labeled donkey anti-rabbit-A546 (A10040), donkey anti-mouse-A647 (A31570) and donkey anti-goat-A488 (A11055; all from Invitrogen, Life Technologies, Carlsbad, USA) were incubated at room temperature for 1 h. Slides were mounted using VectaShield mounting medium containing DAPI (Vector Laboratories, Burlingame, USA). For negative controls, the primary antibodies were omitted and substituted with antibodies of the same isotype class with an unknown specificity.

Whole mounts were prepared as previously described [15 (link)]. Mammary tumors were fixed in formalin and embedded in paraffin. hematoxylin and eosin staining (H&E) was used to assess histology. Infiltration by CD3+ and perforin-positive cells was determined through immunohistochemistry with anti-CD3 (Ab828; Abcam, Cambridge, UK) and anti-perforin (Ab16074, Abcam) antibodies as previously described [16 (link)]. Immunostaining was developed using alkaline phosphatase-conjugated streptavidin (Thermo Fisher Scientific, Waltham, MA, USA) and vulcan fast red chromogen (Biocare Medical, Concord, CA, USA). Slides were counterstained with hematoxylin (BioOptica, Milan, Italy) and images were acquired by Leica DMRD optical microscope (Leica, Milton Keynes, UK).

The immunohistochemistry was carried out on formalin-fixed caeca collected at the hatching day. The specimens were processed, embedded in paraffin, and the tissue sections were cut at 4μm. The sections were treated with 10 mL MoL Tris buffer and 1 mL MoL ethylene-diamine tetra-acetic acid, pH 9.0 for 20 min at 90 °C. The block of endogenous peroxidase was done by incubation the sections with 3% H2O2, followed by preincubation overnight at 4 °C in 1% bovine serum albumin in PBS. The sections were stained for 30 min at 37 °C, using the following antibodies that showed reactivity in avian species: a rabbit polyclonal anti-CD3 (1:200; Abcam, Cambridge, UK, ab828), a mouse polyclonal anti-CD20 (1:200; Abcam, Cambridge, UK, ab88247), a rabbit polyclonal anti-S100 protein antibody (1:200, Thermo Fisher Scientific, Cat. RB-044-A0), and a rabbit polyclonal anti-SIgA (1:400; Abcam, Cambridge, UK, ab2411), according to the method described by Hsu and colleagues^{28 (link)}. The sections were counterstained with haematoxylin, and analysed using an Olympus microscope (Japan). In parallel, tissue specimens, in which the primary antibodies were omitted and replaced with buffer, served as negative controls.

The following polyclonal or monoclonal antibodies (mAbs) were used for immunohistology: anti-CD3 (for rats: clone 1F4, Bio-Rad, Oxford, UK; for dogs: Ab828, Abcam, Cambridge, UK; for minipigs: clone 8E6, WSU Monoclonal antibody center, Pullman, WA; for monkeys: clone CD3-12, Abcam), anti-CD4 (for rats: clone OX-35, Bio-Rad; for dogs: clone DH-29A, WSU Monoclonal antibody center; for minipigs: clone 74-12-4, WSU Monoclonal antibody center; for monkeys: clone BC/1F6, Abcam), anti-CD163 (for all species: clone AM-3K, Antibodies online, Paris, France), anti-CD172a (for rats: clone ED9, Bio-Rad; for dogs: clone DG-DH59B, WSU Monoclonal antibody center; for minipigs: clone BL1H7, Bio-Rad; for monkeys: Ab139698, Abcam), anti-MHC-II (for rats: clone OX-6, Bio-Rad; for dogs: clone DG-H42A, WSU Monoclonal antibody center; for minipigs: clone TH21A, WSU Monoclonal antibody center; for monkeys: clone L243, Abcam). Corresponding isotype-matched mAbs were used as controls in all experiments.