Alexa fluor 488 conjugated goat anti mouse igg

Alpha-SMA (alpha smooth muscle actin, α-SMA) and ALP (alkaline phosphatase) are markers for myofibroblasts and pre-osteoblasts, respectively [51 (link)]. After 1, 7, and 14 d, cellular differentiation of HPLC cells in collagen scaffolds was explored by α-SMA and ALP immunofluorescent staining. Briefly, HPLF-laden collagen scaffolds were fixed with 4% paraformaldehyde in 0.1 M PB, pH 7.4, for 1 min and then blocked with 5% BSA for 30 min at room temperature. Samples were incubated with Recombinant Alexa Fluor^® 647 anti-α-SMA antibody (Abcam, Cat. No. ab196919, 1:100) or anti-ALP antibody (Abcam, Cat. No. ab126820, 1:100) in 1% BSA in PBS in a humidified chamber for 1 h at room temperature in the dark. Thereafter, the samples were incubated with a goat Anti-Mouse IgG-conjugated Alexa Fluor^® 488 (Abcam, Cat. No. ab150113) in 1% BSA in PBS for 1 h at room temperature in the dark. Finally, mounting medium with DAPI (Abcam, Cat. No. ab104139) was applied directly on top of the specimen and covered with coverslips. The expression of α-SMA and ALP was observed under the Dragonfly Confocal Microscopy System (Oxford, Andor) and conducted using an image analysis software (Image J, v. 1.53 t) [52 (link)].

After removing culture medium and rinsing with phosphate-buffered saline (PBS), cell cultures were fixed with 4% formaldehyde in PBS and blocked with normal goat serum (Cell Signaling Technology, Danvers, MA). HSP60 and RTPOR immunoreactivity in cell cultures were probed using HSP60 (#ab3080, Abcam Cambridge, MA) and RPTOR antibodies (#2280, Cell Signaling Technology, Danvers, MA). Goat anti-mouse IgG conjugated Alexa Fluor^® 488 and donkey anti-mouse IgG conjugated Alexa Fluor^® 675 (Abcam, Cambridge, MA) were used as secondary antibodies. After counterstaining with 4′,6-diamidino-2-phenylindole (DAPI) and covering with Prolong Gold Antifade Reagent (Cell Signaling Technology, Danvers, MA), immunofluorescence was evaluated using an Olympus Laser Confocal Microscope system. In some experiments, autophagic puncta in cell cultures were detected using fluorescent monodansylcadaverin probe of Autophagy Detection Kits (Abcam, Cambridge, MA), according to the manufacturer’s instructions. Number of puncta in 30 cells in three random fields of each well were counted.

Cross-sections of carotid arteries were used for confocal microscopic imaging. Platelets were stained with antibodies against CD42d (AF6990, R&D, USA), CD41 (553847, BD, USA) and PF4 (ab282111, Abcam, USA). Monocytes/macrophages were stained with rat anti-mouse F4/80 antibody (MAB5580, R&D, USA). Neutrophils were stained with Ly6G antibody (sc-52515, SANTA CRUZ, USA). Endothelial cells were stained with rabbit anti-mouse CD31 (ab28364, Abcam, USA). Podoplanin expression was determined with Syrian Hamster anti-mouse podoplanin antibody (14-5381-82, eBioscience, USA). Cytoskeletal proteins were determined with phalloidin antibody (CA1610, Solarbio, China). Secondary antibodies Alexa Fluor 488-conjugated donkey anti-rat IgG, Alexa Fluor 488-conjugated goat anti-mouse IgG, Alexa Fluor 647-conjugated donkey anti-rabbit IgG, and Alexa Fluor 647-conjugated goat anti-Syrian hamster IgG were all from Abcam. Donkey anti-sheep IgG NorthernLights™ NL557-conjugated antibody was purchased from R&D. Tissues were counterstained with DAPI before being mounted and examined by confocal microscopy, an Olympus multiphoton laser scanning microscope with high S/N ratio objectives and suppressed autofluorescence, which enables advanced deeper imaging with high resolution (FV1000MPE and FV3000MPE Olympus, Japan).