Tbst rinse buffer

Tumors were harvested and fixed in 10% formalin (Fisher Scientific) for 24 h, then transferred to 70% ethanol. Further studies were performed post radioactivity decay. A 70–100% EtOH gradient was used to dehydrate the tissues over 12 h prior to clearance with histology-grade xylene (Leica) and paraffin embedding (Paraplast Plus, Leica). Next, the tissues were sectioned into 4 µm slices with a Leica RM2235 microtome, transferred onto Superfrost Plus slides (Fisher Scientific), and baked for 30–60 min at 60 °C. Antigen retrieval was performed using citrate and EDTA retrieval solutions at pH 6 and 9, respectively, and a decloaking chamber set to 120 °C. The slides were stained using an Autostainer Plus (Dako) with TBST rinse buffer (Dako). A CD3 antibody (polyclonal, Biocare Medical) was applied using a 1:400 dilution, while the TIGIT antibody (clone EPR26037-152, Abcam) was applied using a 1:100 dilution. For CD3 and TIGIT detection, a Mach 2 Rabbit polymer (Biocare Medical) and a Boost Rabbit HRP polymer (Cell Signaling, Danvers, MA) were used. The substrate used was 3,3, Diaminobenzidine + (Dako). The slides were then counterstained with Hematoxylin (Dako).

Tumor tissue from three patients identified through TIES was available for histological analysis. Formalin Fixed, Paraffin Embedded (FFPE) paired primary tumor and OM tissue was available from one patient; only OM tissue was available for the other two patients. Tissue sections were cut (4 μm) and stained, one with hematoxylin and eosin (H&E), one with an E-cadherin antibody, and one with an estrogen receptor (ER) antibody. For antibody staining, the slides were deparaffinized, rehydrated, and stained using a standard histology protocol. Antigen retrieval was performed using a citrate buffer (Dako, Carpinteria, CA) in a decloaking chamber at 123 °C before being stained using an Autostainer Plus (Dako) platform with TBST rinse buffer (Dako). The E-cadherin antibody (Mouse monoclonal – 4A2C7, Invitrogen, Carlsbad, CA) was applied using a 1:500 dilution at room temperature followed by a secondary antibody of Mach 2 Mouse HRP (Biocare Medical, Pacheco, CA). The ER antibody (Mouse monoclonal – 1D5, Dako) was applied using a 1:100 dilution at room temperature followed by a secondary antibody anti-mouse HiDef HRP Polymer System (Cell Marque, Rocklin, CA). Pictures were taken using a × 200 magnification with the software SPOT imaging.

Mouse tissues were fixed in 10% neutral buffered formalin (Sigma) and embedded in paraffin, then sectioned (5 μm) onto glass slides. Slides were deparaffinized and rehydrated using a standard histology protocol. Antigen retrieval was performed using 10 mM sodium citrate pH 6.0 buffer and a Decloaking chamber at 120°C. The slides were stained using an Autostainer Plus (Dako) with TBST rinse buffer (Dako). The following antibodies were used for IHC: anti-CD4 (Cell Signaling, D7D2Z, 1:100), anti-CD8 (Cell Signaling, D4W2Z, 1:400), anti-CD3 (Biocare Medical, 1:100), and anti-CD19 (Cell Signaling, D4V4B, 1:800). Detection consisted of Rabbit Boost (Cell Signaling) HRP polymer used with 3,3’-diaminobenzidine (DAB) (Dako). Slides were finally counterstained with hematoxylin (Dako). Images were captured with an Olympus AX70 microscope using the Q-Capture Pro7 Program. H&E stained sections and immunohistochemistry of lymphoid malignancies and polycystic changes were evaluated by two board-certified pathologists including a veterinary pathologist (LHR).