Rabbit anti mouse cd206 antibody

Mouse PBMNCs were cultured in chamber slides (Millipore Millicell EZ slide; Merck, Darmstadt, Germany) at a density of 2.5 million cells/mL of 5G-culturemedium/well. After 5 days, E-MNCs were incubated with rat anti-mouse CD11b antibody (1:100; Abcam, Cambridge, MA, USA), rabbit anti-mouse CD206 antibody (1:100; Abcam), rabbit-anti mouse Msr1 antibody (1:200; Bioss, Woburn, MA, USA), or rabbit anti-mouse IGF1 antibody (1:200; Bioss). As secondary antibodies, Alexa Fluor 546–conjugated goat anti-rabbit antibody (1:200; Thermo Fisher Scientific Life Sciences, Waltham, MA, USA) for CD206, Msr1, and IGF1, and Alexa Fluor 647–conjugated goat anti-rat antibody (1:200; Cell Signaling Technology, Danvers, MA, USA) for CD11b were used. Mounting medium for fluorescence with 4′,6-diamidino-2′-phenylindole (DAPI; 1:1500; DOJINDO, Kumamoto, Japan) was employed for nuclear staining.

The RAW264.7 cells, which had undergone different treatments, were washed three times with PBS for 3 min each time. They were then fixed with 4% paraformaldehyde for 15 min, followed by three additional PBS washes, each for 3 min. Permeabilization was achieved by incubating the cells with 0.5% Triton X‐100 at room temperature for 20 min, followed by another PBS wash. The blocking step was carried out by adding 5% BSA at room temperature for 30 min. After removing the blocking solution, an appropriate amount of either rabbit anti‐mouse CCR7 antibody (1:100) or rabbit anti‐mouse CD206 antibody (1:50, Abcam) was added, and the cells were incubated at 4 °C overnight. The following day, the primary antibodies were removed, and Alexa Fluor 488‐labeled goat anti‐rabbit antibody (1:500, Abcam) was added to bind to CCR7, along with Alexa Fluor 594‐labeled goat anti‐rabbit antibody (1:500, Abcam) to bind to CD206. After a 1‐h incubation, the cells were washed three times with PBS, and DAPI was added for light‐protected incubation for 5 min. CLSM was used for image acquisition.

Lipofectamine 2000 (lipo) and pUNO1-mIL12 encoding IL-12 (pIL-12) were obtained from Invitrogen. siRNA targeting HBx (sense: 5′AACGACCGACCUUGA- GGCAUATT3′, antisense: 5′UAUGCCUCAAGGUCGGUCGUUTT3′) [32 (link)] was provided by Sangon Biotech Co., Ltd. (Shanghai, China). Rabbit anti-mouse CD80 antibody, mouse anti-human CD47 antibody, and rabbit anti-human CD80 antibody were obtained from Wuhan Mitaka Biotechnology Co. Ltd. (Wuhan, China). Rabbit anti-human p-PI3K, PI3K, IPS-1, RIG-1, and Bcl-2 antibodies, mouse anti-human p53 antibody, mouse anti-human HLA-1 antibody, and rabbit anti-mouse CD206 antibody were supplied by Abcam. Rabbit anti-human p-ERK, ERK, p-Akt, Akt, and PD-L1 antibodies were supplied by CST. ELISA kits for the detection of mouse IL-12, mouse IL-6, and mouse TNF-α were from 4A Biotech Co., Ltd. (Beijing, China). The ELISA kit for the detection of HBsAg was obtained from Huijia Biotech Co., Ltd. (Xiamen, China).