Anti human vimentin

All samples (pellets and distal femurs) were fixed in 4% paraformaldehyde and embedded in paraffin wax, followed by dehydration with an ethanol series and clearance with xylene. To evaluate osteochondral tissues, the dissected femoral ends were decalcified with 10% ethylenediaminetetraacetic acid (pH 7.4) before paraffin embedding. The samples were cut into 5-µm-thick sections and used for HE staining, toluidine blue staining, Saf-O staining, and immunohistochemistry, as described previously [51 (link),53 (link)]. The following antibodies were used: anti-COL2 (Kyowa Pharma Chemical, Toyama, Japan, Cat# F-57, 1:500 dilution), anti-COL10 (Thermo Fisher Scientific, Cat# 14-9771-82, 1:500 dilution), and anti-human vimentin (Abcam, Cambridge, UK, Cat# ab16700, 1:100 dilution). The CSA of pellets and COL2 staining were measured automatically using an Aperio Image Scope (Leica Biosystems, Wetzlar, Germany). The sections stained with HE and Saf-O were used for histological evaluation using the O’Driscoll scoring system for cartilage repair, as described previously [54 (link)].

The fat graft and PRP were obtained from a consenting human volunteer undergoing liposuction for lipodystrophy. Institutional ethical committee approval was obtained. Alginic acid sodium salt from brown algae (4–12 cP, Low viscosity), pectin derived from citrus peel (Galacturonic acid > 74%), calcium chloride, Oil Red O, Harri’s Hematoxylin, and Eosin solution were procured from Sigma. Anti-human CD 31, anti-human perilipin, and anti-human vimentin antibodies were purchased from Abcam. α-MEM, Pen-strep, FBS, and Trypsin-EDTA were obtained from Invitrogen. Live staining kit and Quanti-iT Picogreen dsDNA assay kit were procured from Thermo Fisher Scientific.

Metformin, 17β-estradiol and Compound C were purchased from Sigma-Aldrich (St. Louis, MO, USA). The anti-human E-cadherin, anti-human N-cadherin, anti-human Vimentin, and anti-human βKlotho primary antibodies were purchased from Abcam (Cambridge, MA, USA). The anti-human ERK1/2, anti-human phospho-ERK1/2 (Thr202/Tyr204), anti-human AMPKα, anti-human phospho-AMPKα (Thr172), anti-human Slug, and anti-human Snail primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-human β-actin and anti-human GAPDH primary antibodies and HRP-conjugated goat anti-rabbit and HRP-conjugated goat anti-mouse secondary antibodies were purchased from ZSGB-BIO (Beijing, China).