Rabbit anti mouse cd4

Duodenum samples were fixed with 4% paraformaldehyde and embedded in paraffin. Paraffin sections (4-5 μm) were deparaffinized, rehydrated, and underwent antigen retrieval. The sections were incubated with rabbit anti-mouse CD4 (Abcam) and rat anti-mouse Foxp3 (eBioscience) antibodies after blocking. Secondary antibodies, FITC-labelled goat anti-rat Ab for Foxp3, and Cy3 goat anti-rabbit Ab for CD4 were used. The signal was detected using the Olympus Provis fluorescence microscope (Nikon Eclipse Ti-SR) with 200× magnification. The mean fluorescence intensity (MFI) was calculated using Image-Pro Plus 6.0.

Rehydrated paraffin-embedded lung sections were treated with Antigen Unmasking Solution according the manufacturer’s instruction (Vector Laboratories Inc. Burlingame, CA, USA) to unmask the antigen. After blocking with 1% bovine serum albumin, the sections were incubated with mouse anti-influenza NP, rabbit anti-mouse CD4, or rabbit anti-mouse CD8 primary antibody (all from Abcam) at 4 °C overnight^{39 (link)}, followed by biotin-conjugated secondary antibody (Calbiochem, Darmstadt, Germany) for 30 min at room temperature. Streptavidin peroxidase complex reagent (Vector Laboratories, Burlingame, CA) was then incubated at room temperature for 30 min followed by color development with 3, 3′-diaminobenzidine (DAB, Vector Laboratories). The slides were mounted and examined under microscope. Representative images were captured with Olympus BX53 semi-motorized fluorescence microscope.

The following primary antibodies were used for IHC: rat anti-mouse IDO1 (1:1000, Cat# 122402) from BioLegend; rabbit anti-mouse CD8 (1:200, Cat# ab203035), rabbit anti-mouse CD4 (1:500, Cat# ab183685), rabbit anti-NCR1 (1:200, Cat# ab214468) and rabbit anti-Ki67 (1:400, Cat# ab21700) from Abcam; rabbit anti-CD31 (1:100, Cat# 77699) and rabbit anti-human IDO1 (1:100, Cat# 86630) from Cell Signaling Technology (CST). Slides were incubated with primary antibodies at 4 °C overnight. The remaining steps were performed according to horseradish peroxidase-conjugated secondary antibody instructions (Cat# PV-9004 or PV-9001, Zhongshan Jinqiao Biotechnology Co., Ltd. China). The slides were developed with diaminobenzidine (DAKO) and counterstained with hematoxylin (Sigma). TUNEL staining in tumor tissues was detected using a TUNEL-POD kit (Cat# KGA7052, KeyGen Biotech, China) according to the manufacturer’s instructions. Positively stained cells in 5 randomly selected fields were counted, and the mean number of positive cells per field was calculated. Staining of IDO1, CD8 and CD4 (T cell marker panels), NCR1 (a pan NK marker), CD31 (a tumor microvessel marker), Ki67 (a proliferation marker) and TUNEL (an apoptosis marker) was quantitatively analyzed by Image-Pro Plus 6.0.