Anti f4 80

Antibodies were obtained from multiple sources: anti-GAPDH from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-MBD2, anti-collagen I, anti-α-SMA and anti-fibronectin from Abcam (Cambridge, MA, USA), and anti-F4/80, anti-collagen IV, and anti-G0S2 from Proteintech Group (Rosemont, IL, USA). The recombinant human TGF-β1 was obtained from Proteintech Group. The plasmids containing the methylation promoter of the G0S2 CpG-free pCpGI luciferase reporter, MBD2, and mtMBD2 (the deletion of the methylated DNA binding domain), were constructed by the Ruqi Biology Company (Guangdong, Guangzhou, China) according to previously published reports [15 (link), 17 (link)]. The MBD2 siRNA was designed and synthesized by the Ruibo Biology Company (Guangdong, Guangzhou, China) as described in our previously published paper [18 (link)].

Primary antibodies for NPM/B23 (ab10530), DNA-PKcs (ab70250), phospho-ATR (T1989) (ab227851), gamma γH2AX (S139) (ab26350), and phospho-ATM (S794) (ab119799) were obtained from Abcam (Cambridge, UK). Antibodies for phospho-p53 (Ser15) (#9284), phospho-ATM/ATR substrates (S/T*Q motif) (#9607), and MMP-2 (#40994) were from Cell Signaling (Beverley, MA, USA). Anti-MMP-9 was from R&D Systems (AF909) (Minneapolis, MN, USA). Anti-F4/80 was from Proteintech Group (28463-1-AP) (Wuhan, China). For short hairpin RNA (shRNA)-mediated gene silencing, 3 different constructs were designed and synthesized by Genepharma (Shanghai, China). The sequence showing the highest efficiency (5′-GCACAGACTGTCTTCCTTA -3′) was further cloned into pGLVU6/GFP lentiviral vector. Lentivirus packaging and purification were provided by Genepharma.

Deeply anesthetized mice were transcardially perfused using 4% paraformaldehyde in phosphate-buffered saline (PBS). Gut tissue was removed and post-fixed overnight in 4% paraformaldehyde at 4 ℃ and equilibrated in 30% sucrose. Frozen 20 μm coronal colon sections were hydrated, immersed in 3% hydrogen peroxide in methanol, and incubated for 1 h with 5% normal goat serum at RT. Next, in a humidified chamber, mouse anti-Ly6G (1:200, Abcam), anti-CD3 (1:200, Novusbio), and anti-F4/80 (1:200, Proteintech) antibodies were incubated with sections overnight at 4 ℃. After washing in PBS, Ly6G, CD3, and F4/80IHC localization were determined using a HRP/DAB kit (Catalog number: 20774 and 20775, MilliporeSigma). Images were captured as described. We quantified immunostaining using ImageJ (NIH). For all sections, the same threshold settings and image exposure times were used.