Rat anti f4 80 clone bm8

The following primary antibodies were used for immunostaining of optically cleared tissues: polyclonal rabbit anti‐α‐smooth muscle actin (SMA) (Abcam; ab5694; 1 : 300); and from (BioLegend UK Ltd, London, UK): rat anti‐CD45 clone 30‐F11 (103102; 1 : 300), CD11c clone N418 (117302; 1 : 200) and MHCII I‐A/I‐E clone M5/114.15.2 (107601; 1 : 300). The following macrophage markers were found to be unreliable with the tissue clearing protocols used: rat anti‐F4/80 (clone BM8), rat anti‐CD11b (clone M1/70), rat anti‐CD68 (clone FA‐11) (all from BioLegend) and rat anti‐F4/80 (clone Cl:A3‐1; from AbD Serotec, Kidlington, UK). The following secondary antibodies (all used at 1 : 500) were purchased from Invitrogen: goat anti‐rabbit Alexa Fluor 488 (A11008), goat anti‐rat Alexa Fluor 647 (A21247) and goat anti‐rat Cy3 (A10522); and from (Jackson ImmunoResearch, Ely, UK): goat anti‐Armenian hamster Cy3 (127‐165‐160).
For 2D analysis, SMA expression was detected using a mouse anti‐human SMA primary antibody (clone 1A4, Agilent; M0851) with a peroxidase‐conjugated ImmPRESS anti‐mouse IgG polymer detection kit (Vector Laboratories Ltd; MP‐7402, Peterborough, UK) using standard development with DAB. Mouse IgG1 was used for species‐ and isotype‐matched control (Agilent; X0931).

Tissues were fixed in 4% (w/v) paraformaldehyde at 4 °C overnight and embedded in O.C.T. compound (Sakura Finetek, Tokyo, Japan). To analyze the localization of macrophages, immunohistochemistry was performed on frozen tissue sections (10 μm). Specifically, sections were treated with REAL Target Retrieval Solution (DAKO, Carpinteria, CA, USA) at 98 °C for 40 min to retrieve the antigen and incubated in 3% H₂O₂ at room temperature (RT) for 10 min to inhibit endogenous peroxidase. After washing, sections were incubated with 0.5% (w/v) blocking reagent (PerkinElmer, Waltham, MA, USA) at RT for 30 min to block the non-specific binding of antibodies and then treated with primary antibodies at 4 °C overnight followed by secondary antibodies at RT for 1 h. The primary antibodies used in the present study were rabbit anti-CD11c (clone D1V9Y, 1:100, Cell Signaling Technology, Danvers, MA, USA) and rat anti-F4/80 (clone BM8, 1:50, BioLegend). Secondary antibodies were horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG (1:2000, Jackson ImmunoResearch, West Grove, PA, USA) and Alexa Fluor 647-conjugated donkey anti-rat IgG (1:200, Jackson ImmunoResearch). The enzyme activity of HRP was visualized using the TSA Plus fluorescein system (PerkinElmer). Sections were then counterstained with DAPI. Tissue images were obtained using a BZ-9000 microscope (Keyence, Osaka Japan).