Col f

To enable cell response to injury to be visualised in 3D, PFA-fixed tendons (n = 2 per time point) were immunolabelled and optically cleared using HISTO™ solutions (Visikol, Hampton, NJ, USA) according to our recently developed protocol [11 (link)]. Briefly, following permeabilisation and blocking, tendons were incubated with primary antibodies for CD146 (1:100; Abcam: ab75769, Cambridge, UK) for 96 h to allow for the complete diffusion of the antibodies through the full thickness of the tendon. Secondary antibody incubation (Alexa Fluor^® 594 Goat anti-rabbit IgG, Invitrogen, 1:500, Waltham, MA, USA) was performed for 96 h, followed by overnight nuclei counterstaining with DAPI. Some tendons were additionally stained with Col-F (20 µm; ImmunoChemistry Technologies, Bloomington, MN, USA), a fluorescent probe that binds collagen and elastin, during the nuclei counterstaining step to allow for the visualisation of collagen within the tendon in 3D [87 (link)]. Tendons were then washed, dehydrated in methanol and cleared by incubation in HISTO™-1 and -2 solutions for at least 72 h. Tendons were stored in HISTO™-2 prior to imaging.

Bleomycin chlorate was purchased from Nippon Kayaku Company (Tokyo, Japan). Formamide was purchased from Fujifilm Wako Pure Chemicals Co., Ltd. (Osaka, Japan). Polyethylene glycol (8 kDa) was purchased from Sigma Aldrich Co. (St. Louis, MO, USA). DyLight 488-conjugated tomato lectin was purchased from Vector Laboratories Inc. (Burlingame, CA, USA). Col-F was purchased from ImmunoChemistry Technologies LLC (Bloomington, MN, USA). Male 5-week-old ICR mice weighing 26–28 g were purchased from Japan SLC (Shizuoka, Japan). The Laboratory Animal Center approved the animal experimental protocol (No. H29-001), which conformed to the Guiding Principles for the Care and Use of Experimental Animals at Hokkaido Pharmaceutical University.