Cd8 clone sp16

CRC tissues were fixed using neutral-buffered formalin and embedded in paraffin. The sections were stained with Perls’ reagent and developed using DAB, as previously described (42 (link)). After iron staining, the slides were subsequently incubated with primary antibodies against CD8 (clone SP16, ab9829; Abcam), CD66b (clone G10F5, 555723; BD Pharmingen), cytokeratin 20 (clone Ks20.8, 413491; Nichirei), and IBA1 (polyclonal, 019-19741; Wako) overnight at 4°C. The sections were visualized using HistoGreen (E109; Cosmo Bio) and counterstained with Mayer hematoxylin. CCL8 (clone 1.1_2D4-1A3, LS-B8198; LSBio) staining was conducted using DAB, followed by costaining with IBA1 using HistoGreen. Images were obtained with a KEYENCE BZ-X800 all-in-one microscope (KEYENCE). Quantification was performed using the KEYENCE BZ analyzer.

IDH1 mutation status and MGMT expression was tested post-hoc by immunohistochemistry of biopsy specimens obtained immediately prior to the first G47∆ injection. Formalin-fixed, paraffin-embedded tissue sections of 3-μm thickness were deparaffinized in xylene and rehydrated in a series of graded alcohols. Antigen retrieval was performed with citrate buffer pH6 for CD4, CD8, and MGM and EDTA buffer pH9 for HSV.
Endogenous peroxidase was blocked by incubation with 3% hydrogen peroxidase for 5 min at room temperature. Then sections were incubated with the following primary antibodies: CD4 (clone EPR6855, dilution 1:250, Abcam); CD8 (clone SP16, dilution 1:100, Abcam); HSV (polyclonal, ready-to-use, Gene Tex); IDH1 R132H (clone H09, dilution 1:100, Dianova); MGMT (clone MT3.1, dilution 1:100, Abcam) for 30 min at 37 °C, respectively. After washing, sections were treated with secondary antibody (Envision + Dual Link System-HRP, Dako) for 30 min at 37 °C. To visualize the antigen-antibody complex, ImmPACT DAB substrate kit (Vector Laboratories) was used, and then sections were counterstained with hematoxylin. Supplementary Table 5 provides details on primary antibodies and antigen retrieval.