Alexa fluor 488 or 594 conjugated donkey igg

Cells were fixed in −20 °C cold methanol for 10 minutes then blocked with phosphate buffered saline containing 5% donkey serum at room temperature for 30 minutes. For sectioning of 3D collagen 3T3-L1, collagen plug was embedded in Optimal Cutting Temperature compound (Sakura Finetek USA, Torrance, CA) as previously described (Lee et al., 2004 (link)). Sectioning was performed at University of California–San Diego Moores Histology Core (La Jolla, CA). The detailed immunostaining method has been described (Sato et al., 2016 (link)). Anti-pCREB, anti-perilipin, anti-decorin, and Alexa Fluor 488- or 594-conjugated donkey IgG (Thermo Fisher Scientific) were used at 1μg/ml. Cover slips were mounted using ProLong Gold Antifade Reagent with DAPI (Thermo Fisher Scientific). Images were captured using a BX41 microscope (Olympus, Center Valley, PA).

TIP39 peptides were obtained from Bachem Americas (Torrance, CA). Goat anti-decorin (R&D, Minneapolis, MN), rabbit anti-CREB (Abcam, Cambridge, MA), rabbit anti-pCREB (Abcam), rabbit anti-laminB1 (Abcam), goat anti-perilipin (Abcam), goat anti-actin (Santa Cruz, Dallas, TX), and rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (Abcam) were used as primary antibodies for immunoblotting or immunostaining. Alexa Fluor 488- or 594-conjugated donkey IgG (Thermo Fisher Scientific, Waltham, MA) were used as secondary antibodies for immunostaining.