Anti mouse cd4 antibody

Another set of mice was injected with 2 × 10⁶ MBT-2 and treated by the same method described above. Tumors were resected and divided into two pieces, and half was fixed with 4% paraformaldehyde-PBS and embedded in paraffin. Another piece of tumor was used for flow cytometry as described below. Immunohistochemical staining was performed as in our previous study.²⁵ (link) Anti-mouse CD4 antibody (1:1,000, Abcam, Cambridge, UK) and anti-mouse CD8a antibody (1:400, Cell Signaling Technology Japan, Tokyo, Japan) were used in the immunohistochemical staining. The tissue slides were observed with a BZ-X710 microscope (Keyence, Osaka, Japan).

The following monoclonal antibodies (mAbs) were purchased from BioLegend, BD Bioscience and Abcam: antimouse CD8a antibody (clone 53–6.7, PE/Cyanine7 conjugated), antimouse CD4 antibody (clone GK1.5, FITC conjugated), antimouse CD45R/B220 antibody (clone RA3-6B2, FITC conjugated), anti-H2AX (pS139) antibody (clone N1-431, PE-conjugated) and anti-gamma H2AX (pS139) antibody (clone EP854(2)Y, Alexa Fluor® 488-conjugated). Cell surface antigen staining was performed for FCM only. Subsequently, we used a fixation and permeabilization solution to permeate the membrane and conduct intracytoplasmic staining using the aforementioned anti-H2AX (pS139) antibody. The whole staining procedure was conducted per the manufacturer's guidelines and protocols. Stained cells were run on a flow cytometer (RF-500, Sysmex Corporation, Kobe, Japan) for FCM.