3 3 diaminobenzidine substrate

After sample preparation, the 39 sections were washed twice in PBS (Sigma-Aldrich) for 5 min. The sections were blocked with a solution of 10% normal serum (FBS; Gibco, Grand Island, NY) and 1% BSA (Sigma-Aldrich) in PBS for 3 h and then incubated with anti-β2M antibody (ab195531, Abcam, Cambridge, MA) at 1:400 dilution for 30 min. We added 3,3′-diaminobenzidine substrate (Abcam) and incubated the sectioned samples for 5 min, followed by thorough washing with tap water for another 5 min, and finally staining with Mayer’s Hematoxylin Solution for 10 min. The sections were dehydrated with a series of graded alcohol washes and then mounted for evaluation under light microscopy.

Paraffin-embedded sections were deparaffinized by 2 changes of clarene and rehydrated through an alcohol gradient. A heated antigen retrieval with 10 mM citrate buffer pH 6.0 was performed. Samples were blocked with 10% goat serum (Sigma-Aldrich) in phosphate-buffered saline for 30 minutes at room temperature and incubated with the unconjugated primary antibodies (α–smooth muscle actin [SMA], 1:100, Sigma-Aldrich; Isolectin B4-Biotin 1:100, Life Technologies; Elastin, 1:100, Santa Cruz Biotechnology) overnight at 4°C. Fluorophore-conjugated (Alexa Fluor 488 and Alexa Fluor 546, 1:400, Life Technologies) or chromogen-conjugated (horseradish peroxidase, 1:1,000, R &D Systems) secondary antibodies were incubated on the sections for 1 hour at room temperature in the dark. Incubation with 3,3’diaminobenzidine substrate (Abcam) was used for detection of horseradish peroxidase–derived signal. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (1:1000, Sigma-Aldrich) for fluorescent staining, or with hematoxylin for immunohistochemical staining. Slides were mounted with Hardset mounting medium (Vectashield). Images were taken with a Zeiss Observer.Z1 fluorescent microscope. ImageJ software was used to quantify the SMA and Elastin expression in the tissue sections.

Hematoxylin and eosin (H&E) staining was performed as described previously (11 (link)). Briefly, skin tissue was fixed in 10% buffered formalin and embedded in paraffin, then cut into 5-μm sections using a microtome (HM 325, ThermoFisher, Grand Island, NY, USA). At least 3 independent skin tissue sections from each group were H&E stained and examined for histological changes using a Keyence Fluorescence Microscope Model BZ-X710 (Osaka, Japan).
Immunohistochemistry (IHC) was conducted with a rabbit-specific IHC polymer detection kit (Abcam, Cambridge, MA, USA) per the manufacturer’s protocol. The macrophage marker F4/80 was detected using antibody (Cat No. ab100790, Abcam) with 3,3’-diaminobenzidine substrate (Abcam), and slides were examined under the microscope.