Alexa fluor 568 conjugated goat anti rabbit immunoglobulin g

Cells grown on the bottom of culture wells were stained directly. The cells were rinsed briefly in D-PBS and fixed using a sequential paraformaldehyde/methanol fixation procedure {incubation in 4% paraformaldehyde in PBS (20 mM sodium phosphate buffer, pH 7.4, 0.9% NaCl) at room temperature for 10 min followed by incubation in methanol at -20℃ for 20 min}.23) (link) The cells were incubated overnight at 4℃ in preblocking buffer (5% normal goat serum, 0.05% Triton X-100, and 450 mM NaCl in 20 mM sodium phosphate buffer, pH 7.4). Primary antibodies {mouse monoclonal anti-GlcNAc (1:1000; ABR Bioreagents, Golden, CO, USA), and rabbit anti-OGT (O-GlcNAc transferase, 1:500; ProteinTech Group, Inc, Chicago, IL, USA} were added to the wells and incubated overnight at 4℃. The cells were rinsed (3×15 min in preblocking buffer) and incubated with secondary antibodies {Alexa Fluor 488-conjugated goat anti-mouse and Alexa Fluor 568-conjugated goat anti-rabbit immunoglobulin G (each diluted 1:1,000 in blocking buffer; Invitrogen)} at room temperature for 1-2 hr. The cells were rinsed once in preblocking buffer for 15 min, twice in D-PBS, and a mounting solution (4% N-propylgallate in 90% glycerol and 10% sodium carbonate buffer, pH 8.7) was added to cover the bottom of the well.

CFs were analyzed for cardiac α‑SMA expression by immunofluorescence to assess the transformation of CFs into myofibroblasts. The cells on the glass coverslips were washed 3 times with PBS (for 5 minutes each), fixed with 4% paraformaldehyde (10 minutes), permeabilized in 0.2% Triton X‐100 in PBS (10 minutes), and blocked with 10% goat serum for 60 minutes at room temperature before being stained with an anti‑α‑SMA antibody at a dilution of 1:100 with 1% goat serum overnight at 4°C. The cells were washed 5 times with PBS and then incubated with an Alexa Fluor 568‐conjugated goat anti‑rabbit immunoglobulin G (Invitrogen, USA) secondary antibody for 1 hour at 37°C. Finally, the coverslips were washed 5 times and then mounted onto glass slides with DAPI. Immunofluorescence images were obtained with a fluorescence microscope (Olympus DX51, Japan).