Alexa fluor 647 igg

Anti-TSPAN4 (Biossusa, Cat#bs-9413R); anti-TSPAN7 (Biorbyt, Cat#orb373388); anti-integrin β1 (SantaCruz, Cat#sc374429); anti-CXCL12 (Proteintech, Cat#174021AP); anti-CXCR4 (Abcam, Cat#ab124824); anti-CD34 (Abcam, Cat#ab81289); anti-Perilipin (Progen, Cat#Gp40); anti-RhoA (Abcam, Cat#ab86297); anti-PPARγ (Abcam, Cat# ab209350) antibodies; goat anti-rabbit Alexa Fluor® 488 IgG (Abcam, Cat# ab150077); goat anti-guinea pig Alexa Fluor® 647 IgG (Abcam, Cat# ab150187); goat anti-rabbit Alexa Fluor® 647 IgG (Abcam, Cat#ab150079); goat anti-mouse Alexa Fluor® 488 IgG (Abcam, Cat#ab150113); AMD3100 (Abmole, Cat#M1898); CCG-1423 (Abmole, Cat#M8999); culture medium for mouse ASCs (OriCell, Cat#MUXMD9011); PBS (Gibco, Cat#10,010,023); trypsin–EDTA (Gibco, Cat#25,200,056); siRNA targeting CXCL12 (SANTA CRUZ, Cat#sc-39,368).

Full-thickness biopsies of the grafts were obtained before and after grafting. Tissue sections were incubated overnight at 4° C with the primary antibody (rabbit anti-mouse type I collagen; 1:200; Sigma) [44 (link)]. Tissue sections were washed three times with PBS and then incubated at 37° C for 1 h with biotin-labeled rat anti-rabbit IgG (1:200; Invitrogen) [45 (link)]. Signals were observed using the avidin–biotin–horseradish peroxidase detection system. Slides were examined under an Olympus BX51 microscope.
Immunofluorescence staining was performed at 4° C overnight with guinea pig anti-mouse perilipin (1:400; Progen, Heidelberg, Germany) [46 (link)]. Tissue sections were washed three times with PBS and then incubated at 37° C for 1 h with rhodamine-conjugated goat anti-guinea pig Alexa Fluor 647 IgG (Abcam, Cambridge, MA, USA) [45 (link)]. Nuclei were stained with DAPI (1:200; Sigma). Images were acquired and analyzed under a C1Si confocal laser scanning microscope (Nikon, Tokyo, Japan).

The location and expression of LC3 in U2OS cells were detected by IF. Briefly, U2OS cells that were seeded in 12-well plates were stained with Mito-Tracker Green solution (Beyotime; Catalog No: C1048) for 30 min. After being washed with phosphate-buffered saline (Beyotime; Catalog No: C0221A), cells were fixed in 3% methanol (Sinopharm; Catalog No: 80080418), treated with 1% Triton-X 100 (Solarbio, Catalog No: T8200), and blocked with 3% bovine serum albumin (Sigma-Aldrich, St Louis, MO, USA; Catalog No: A7906). Subsequently, cells underwent 12 h of incubation with anti-LC3 (1:250, Abcam, Cambridge, UK; Catalog No: ab192890) at 4 °C, and further underwent 1 h of incubation with Alexa Fluor® 647-IgG (1: 500, Abcam; Catalog No: ab150079) and 4′, 6-diamidino-2-phenylindole (DAPI) (Beyotime; Catalog No: C1005) at 25 ℃. After quenching and sealing, cells displaying fluorescence were observed under a microscope (CKX53, Olympus).