Alexa fluor conjugated goat anti mouse igg

In order to identify the stemness of encapsulated MSCs in different hydrogel formulations, immunoflurescence staining was performed using anti-CD146 antibody (Abcam). Briefly, after two weeks of culturing in osteogenic media, the specimens were retrieved, PFA (4%) fixed, paraffin embedded, sectioned (6 μm), and deparaffinized. The slides were treated with 3% H₂O₂ and then with a blocking buffer (1% BSA and 0.25% Triton X-100 in PBS) and stained with CD146 primary antibody (1:200, Abcam) over night. Next, secondary Alexa-Fluor conjugated goat anti-mouse IgG (1:200, Invitrogen) was used and specimens were counterstained with DAPI (Vector Laboratories, Burlingame, CA).

Optima LC/MS solvents (chloroform, methanol, methylene chloride, and water), formic acid (Optima LC/MS), and sodium chloride were purchased from Thermo Fisher Scientific (Grand Island, NY). Benzyldimethyldodecylammonium chloride (BAC C12) and benzyldimethylhexadecylammonium chloride (BAC C16) were purchased from Sigma-Aldrich Co. (St. Louis, MO). BAC C12 and BAC C16 were dissolved in DMSO to yield 1, 10, 50, and 100 μM stocks and stored at −80°C. The deuterated (d₇-) sterol standard d₇-7-dehydrocholesterol was prepared as reported previously.^{30 (link)} d₇-Cholesterol was purchased from Avanti Polar Lipids (Alabaster, AL). ¹³C₃-desmosterol and ¹³C₃-lanosterol were purchased from Kerafast (Boston, MA). The primary antibody used in immunocytochemistry was mouse anti-Ki67 at a dilution of 1:200 (BD Pharmingen^™, San Jose, CA) and the secondary antibody used was Alexa Fluor-conjugated goat anti-mouse IgG at a dilution of 1:1000 (Invitrogen ^™, Carlsbad, CA). 4,6-Diamidino-2-phenylindole (DAPI) was purchased from Thermo Fisher Scientific.