Ab54501

Tumors were frozen in optimal cutting temperature compound (Tissue-Tek, SAKURA) before cryosectioning. Tumor sections were stained with specific primary antibodies, including rat anti-mouse Ly6C (128001, BioLegend), rabbit anti-mouse Ly6G (87048, Cell Signaling), rat anti-mouse CD68 (MCA1957CA, Bio-Rad), rat anti-mouse F4/80 (MCA497GA, Bio-Rad), rabbit anti-mouse CD8 (ab217344, Abcam), and rabbit anti-mouse FOXP3 (ab54501, Abcam). Primary antibodies were detected by goat anti-rat IgG conjugated with Alex Fluor 488 (A11006, Invitrogen) or by goat anti-rat IgG conjugated with Alex Fluor 594 (A11012, Invitrogen). The images were analyzed by Image-Pro plus 6.0 software (MediaCyberneticsm Rockville, MD, USA).

Paraffin sections of the harvested grafts were cut at 5-μm thickness. H&E staining was performed using routine techniques.
Sections for immunofluorescence were incubated with anti-CD68 (Abcam, Cambridge, UK; ab125212) overnight, followed by incubation with fluorescently labeled secondary antibodies (AntGene Biotech, Wuhan, China). Nuclei were stained using 4.6-diamidino-2-phenylindole (DAPI; Linaris, Wertheim, Germany). Isotype-matched control antibodies (Abcam, ab172730) served as negative controls.
Immunohistochemistry was performed as described previously^{35 (link)}. Primary antibodies included anti-CD4 antibody (Abcam, ab183685), anti-CD8 antibody (Abcam, ab203035) and anti-Foxp3 antibody (Abcam, ab54501). For negative controls, primary antibodies were omitted.
The severity of allograft vasculopathy was assessed on elastin-stained arteries, using the neointimal (I)/medial (M) ratio^{33 (link)}. The total number of CD4+ , CD8+ , Foxp3+ and CD68+ cells in each section was counted manually by technicians blinded to the sample origin. Three sections from each group were selected for analysis using the IMAGE-PRO PLUS 6.0 software (Media Cybernetics, Inc., Silver Spring, MD). Error bars represent the standard errors of mean (SEM).

For immunocytochemical analysis, the mouse colonic tissues were fixed with 4 % paraformaldehyde in PBS, embedded in paraffin, and cut into tissue sections (5-μm-thick). The tissues were blocked with 1 % bovine serum albumin (BSA, Nacalai tesque) for 60 min at room temperature. The primary antibodies used in the present study included anti-TLR4 antibody (1:200, ab22048, Abcam, Cambridge, UK), anti-NF-κB antibody (1:200, 8242, CST, Danvers, MA, USA), anti-FOXp3 antibody (1:200, ab54501, Abcam, Cambridge, UK), and anti-PCNA antibody (1:100, Santa Cruz Biotechnology, Dallas, TX, USA). Secondary antibodies included goat anti-rabbit IgG (PV-9001, ZSGB-BIO, Peking, China) and rabbit anti-mouse IgG (PV-9005, ZSGB-BIO, Peking, China). Negative controls substituted with non-immunized mouse IgG at the same concentrations as that for each primary antibody were employed. Images were captured using a Leitz Dialux 22 Microscope (Leica Microsystems, Wetzlar, Germany) equipped with a QIcam Fast 1394 camera (QImaging, Surrey, BC, Canada). The images were analyzed using the software, Image Pro Plus 6.0 (Media Cybernetics UK, Marlow, UK). The levels of expression of TLR4, NF-κB, FOXp3, and PCNA were determined by measuring the positively labeled areas in relation to the total area.