Tsa fluorescein system

We incubated 7 μm frozen sections with anti-CD11c (Invitrogen) and secondary antibody (anti-hamster IgG, Southern Biotech, Birmingham, AL, USA), and sequentially incubated using a TSA Fluorescein System (Akoya Biosciences, Marlborough, MA, USA). The sections were also incubated with anti-collagen type 1 (Novotec, Bron, France) and secondary antibody (anti-rabbit IgG Alexa Fluor 594, Abcam). Finally, the sections were incubated with DAPI (Invitrogen). Images were acquired using a BX50 microscope and its imaging system (Olympus, Tokyo, Japan). The positive signal of each co-localized area was selected according to the method of Tolivia et al. (Figure S6) [47 (link)] and analyzed using Adobe Photoshop CS software, version 8.

Next, 7-μm frozen sections were incubated with anti-CD11c (Invitrogen) and secondary antibody (anti-hamster IgG, Southern Biotech, Birmingham, AL, USA), and sequentially incubated with TSA Fluorescein System (Akoya Biosciences, Marlborough, MA, USA). The sections were also incubated with anti-collagen type 1 (Novotec, Bron, France) and secondary antibody (anti-rabbit IgG Alexa Fluor 594, Abcam). Finally, the sections were incubated with DAPI (Invitrogen). Images were acquired using a BX50 microscope and its imaging system (Olympus, Tokyo, Japan). The positive signal of each colocalized area was selected according to the method of Tolivia et al. (Figure S10) [63 (link)] and analyzed using Adobe Photoshop CS software, version 8.