Rat anti cd4 gk1.5

Unfixed thymi were embedded in OCT compound (Sakura, Chuo-ku, Tokyo, Japan), cut, fixed in acetone and stained as previously described.^{49 (link)} The primary Abs used were rabbit anti-FADD (EP887Y), rabbit anti-Notch1 (EP1238Y), rabbit anti-Jagged1 (EPR4290), rabbit anti-Jagged2 (EPR3646) (Epitomics), rabbit anti-NKAP (Sigma), rat anti-CD4 (GK1.5), rat anti-CD8 (YTS169.4), rabbit anti-DL1 (30B11.1(14)), rabbit anti-DL4 (Abcam, Cambridge, MA, USA), rat anti-CD44 (IM7), and biotinylated rat anti-CD25(PC61) (BD Biosciences). Secondary reagents used were Alexa 488-streptavidin, Alexa 488 and Alexa 594-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA). Sections were first blocked for 1 h using 4% goat serum, then labeled with primary Abs followed by the secondary Abs. Nuclear DNA was stained using 4′, 6-diamidino-2-phenylindole (DAPI). Images were then visualized by microscopy (Carl Zeiss Axioplan 2, Göttingen, Germany).

After the mice were killed, tumor tissues were removed aseptically and immediately fixed in 4% formalin at room temperature for 2 days. The fixed tissues were processed through graded concentrations of ethanol and xylene and were then embedded in paraffin wax. Tissue sections of 4‐5 mm were mounted on adhesive glass slides and were stained with HE. Tumor sections were then deparaffinized and treated with 0.08% H₂O2 for 30 minutes to block endogenous peroxidase. Slides were incubated with rat anti‐CD4 (GK1.5; Abcam, Cambridge, MA, USA) or rat anti‐CD8 (2.43; Abcam, Cambridge, MA, USA) at 4°C overnight, followed by incubation with HRP‐conjugated rabbit anti‐rat Ig. Diaminobenzidine was used to develop the staining reaction, and nuclear counterstaining was performed with hematoxylin. Slides were coded and examined by a pathologist who was blinded for the experimental history of the animals.