Alexa flour 488 conjugated secondary antibody

The procedure and protocol of flow cytometric analysis was according to our recent reports [7 (link), 8 (link)]. In detail, peripheral blood sampling at baseline and at 18 h and on day 14 after CLI induction was obtained via cardiac puncture with a 30# needle. Moreover, the BM levels of EPCs were measured at 18 h and on day 14 after CLI induction by needle aspiration and washing from the long bone of the animals. After treatment with red blood cell-lysing buffer, the cells were labeled with appropriate antibodies. Flow cytometric quantification of EPCs through identification of the chosen cell surface markers was performed based on our recent reports [7 (link), 8 (link)]. Briefly, the cells were incubated for 30 minutes with primary antibodies, including PE-conjugated antibodies (against Sca-1 and CD31, BD Biosciences), FITC-conjugated antibody against c-Kit (BD Biosciences), and anti-KDR (NeoMarkers) antibodies which were further recognized by Alexa flour 488-conjugated secondary antibodies (Invitrogen). Isotype-identical antibodies (IgG) served as controls. Flow cytometric analyses were performed by utilizing a fluorescence-activated cell sorter (Beckman Coulter FC500 flow cytometer).

Formalin-fixed, paraffin-embedded, tumor sections (8 μm) were cut with a microtome, mounted on ChemMateTM capillary gap slides (Dako), dried at 60 °C, deparaffinized and hydrated. Hydrogen peroxide (1.5%) in TBS buffer (pH 7.4) was used for blocking endogenous peroxidase, and antigen retrieval was achieved by microwave boiling in TEG buffer (Dako) for 15 min. Sections were blocked with an Fc receptor blocker (BD Bioscience, #553141) and incubated with 1X blocking buffer (2.5% BSA, 0.1% Triton X-100 in PBS). The sections were incubated overnight at 4 °C with anti-myeloperoxidase (R&D System, AF3667, 1:100) in 0.5X blocking buffer. After three washes with PBS, the sections were incubated with Alexaflour488-conjugated secondary antibodies (Invitrogen; A10034 1:150) in 0.5x blocking buffer for 45 min in the dark at room temperature. After two washes with PBS and one with water, sections were counterstained with DAPI (#D1306, Thermo Fisher Scientific) and rinsed in water, and the slides were mounted onto coverslips using mounting media (Invitrogen, #P36930).

Mouse hearts were fixed in 4% paraformaldehyde/PBS on ice for 30 min. They were then embedded in OCT using standard procedure. Cryosections were cut to 6 μm in thickness for immunofluorescence. Tissue sections were washed with PBS and blocked with 10% goat normal serum for 30 min at room temperature. The tissues were subsequently incubated with either primary antibody anti-mouse cardiac Troponin T (1:200, Thermo scientific), anti-mouse αSMA (1:100, Sigma), or anti-mouse PECAM (CD31) (1:100, BD Biosciences), for 1 hour at room temperature. Slides were washed three times in PBS, followed by incubation with Alexa Flour 488 conjugated secondary antibodies (1:500; Invitrogen) for 45 min at room temperature. Sections were counterstained with diamidino-2-phenylindole (DAPI) and photographed under a fluorescence microscope.

iBMECs were seeded at 250,000 cells cm⁻² on borosilicate cover glass slides (coated with fibronectin and collagen IV as described above) and cultured for 2 days using media outlined above. iBMECs were then washed with 1 × PBS, fixed with ice cold methanol for 15 min, and blocked with 10% goat serum (Cell Signaling Technology #5425) or 10% donkey serum (Millipore Sigma #D9663) supplemented with 0.3% Triton X-100 (Millipore Sigma #108643) in PBS for 30 min. Primary antibodies are summarized in Additional file 2: Table S2. Cells were treated with Alexa Flour-647 and Alexa Flour-488 conjugated secondary antibodies (Life Technologies) diluted 1:200 in blocking buffer for 45 min at room temperature. To localize nuclei, cells were treated with 1 μg mL⁻¹ DAPI (ThermoFisher #D1306). Between each step of the staining protocol, monolayers were washed three times with 1XPBS for 5 min. Images were acquired using a 40× magnification objective (Nikon) on an inverted microscope (Nikon Eclipse Ti-E) with illumination provided by an MLC400 monolithic laser combiner (Keysight Technologies). To enable semi-quantitative analysis of protein levels, we normalized fluorescence signal to nuclear signal across at least four biological replicates for each cell source.

Cells were plated onto coverslips in a 24-well plate and transfected with the indicated plasmids (NS3-Flag, 1000 ng, C19orf66-Myc, 250 ng). At 48 h post-transfection, cells were washed once with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde in PBS. Cells were permeabilized with 0.2% Triton X-100 and blocked for 30 min at room temperature with 10% bovine serum albumin (BSA) in PBS, followed by incubation with the primary antibody for 2 h. After three washes with PBS containing 0.1% Tween 20 (PBST), cells were incubated with rhodamine-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) or with Alexa Flour 488-conjugated secondary antibodies (Life Technologies, Grand Island, NY) for 30 min and then incubated with 4-,6-diamidino-2-phenylindole (DAPI) for 5 min. Finally, the coverslips were washed extensively and fixed onto slides. Before the indicated time point, the cells were stained with LysoTracker (L12492, LIFE) for 2 hours. Images were taken under a Zeiss Axio Imager Z2 and a LSM780 confocal microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany).