Vs120 s6 w

Kidney was fixed in 4% para-formaldehyde, and after dewaxing and rehydrating like in H&E staining, sections were treated with 1% Periodic Acid for 30 min, subsequently, sections were stained with PAS for 45 min, and the nuclei were stained by Hematoxylin for 1 min. Sections were imaged using a Virtual Slide Microscope (VS120-S6-W, Olympus, Japan).

Mouse dorsal skin tissues were fixed in 4% (v/v) formaldehyde, embedded in paraffin, sliced into 5-µm sections (HistoCoreBIOCUT; Leica Camera AG, Germany), and subjected to hematoxylin and eosin (H&E) staining (Liu et al., 2022 (link)). Skin thickness and dermal papilla diameters were measured with Slideviewer software (NDP view2; Beijing, China). A TUNEL assay and ki67 immunofluorescence detection were used to evaluate HFs apoptosis and proliferation. For the TUNEL assay, the slides were dewaxed, rehydrated, deproteinized, washed, incubated in TUNEL solution at 37°C for 1 h, and visualized with horseradish peroxidase-conjugated secondary antibody solution. The cell nuclei were stained with hematoxylin. All sections were observed and imaged with a digital pathology scanner (VS120-S6-W; Olympus, Tokyo, Japan). For the ki67 immunofluorescence detection, the rehydrated slides were heated and subjected to the ki67 antigen retrieval solution in the kit. The slides were blocked using a blocking buffer to avoid non-specific hybridization. The ki67 was then hybridized with a specific antibody and detected using a fluorescent reagent. The nuclei were stained with 4′,6-diamidino-2-phenylindole and imaged by fluorescence microscopy (ECLIPSE C1; Nikon Corporation, Tokyo, Japan).

For morphometric analysis, midsagittal sections of the tibias were dewaxed and stained with Alcian blue/hematoxylin solution and orange G solution. After dehydration, clearing, and mounting, images of the sections were captured by a virtual slide system (VS120-S6-W, Olympus, Japan).

After dewaxing and rehydration, sections were treated with 3% H₂O₂ solution for 15 min at 37 °C, followed by the antigen repair with sodium citrate solution for 15 min at 95 °C. The sections were incubated in primary Nrf2 antibody (16396-1-AP, Wuhan Sanying, Wuhan, China, 1:100) overnight at 4 °C. The sections were then incubated in horseradish peroxidase labeled goat anti rabbit IgG (A0208, Beyotime Biotechnology, Shanghai, China) for 40 min. After staining with diaminobenzidine and counterstaining with hematoxylin, the images were captured by by a virtual slide system (VS120-S6-W, Olympus, Japan), and were analyzed with an image Pro Plus 6.0 software (Media Cybernetics, PA, USA).

The cells (passage 2) were grown in a chamber slide to 70% confluence in DMEM. The media was then removed, washed in sterile PBS, immediately fixed with 5% formalin for 30 min and immunofluorescence assay for tendon specific biomarkers including tenomodulin and scleraxis were carried out by following the above-mentioned protocol. The primary antibodies used were anti-goat tenomodulin (sc-49324, Santa Cruz Biotechnology, Inc) and anti-goat scleraxis (sc-87425, Santa Cruz Biotechnology, Inc), and the secondary antibody used was donkey-anti-goat-594. The images were acquired using a fluorescent slide scanner system (VS120-S6-W, Olympus) at 20x magnification and the images were taken and converted to TIF format using OlyVIA Desktop software. DAPI was used to counterstain the nuclei.