Wga cf405s conjugate

Samples were fixed with 4% paraformaldehyde (PFA) at room temperature for 15 min and washed with sodium borohydride (dissolved in PBS). Cells were stained with the following: rhodamine phalloidin (Life Technologies), Alexa-fluor 647 phalloidin (Life Technologies), α/β Tubulin antibody (Cell Signaling, 1:100), Myosin (Life Technologies, 1:100), WGA-CF405S conjugate (Biotium) and Alexa Fluor 647 Conjugate WGA (Life Technologies). Nuclei were counterstained with DAPI (Life Technologies). Paraffin-embedded sections were deparaffinized and antigen retrieval was carried out using sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) at 100 °C for 30 min. Samples were stained with rabbit anti-human CD31 (1:50), Von Willebrand Factor (1:300), Alexa Fluor 647 Conjugate WGA and DAPI (Life Technologies). For studying the effect of pharmacological inhibitors on cytokinesis bridges, MDA-MB-231 cells were treated with a combination of docetaxel (500 pM–50 nM) with cytochalasin D (50 nM) in complete media for 24 h post 6–18 h of serum deprivation. The cells were immunostained with primary rabbit anti-CEP55 antibody (1:250). For visualizing actin, the cells were immunostained with rhodamine phalloidin (Life Technologies-Invitrogen, USA). The imaging was performed using confocal microscopy.

Primary antibodies used on mouse muscle included eMHC (Developmental Studies Hybridoma Bank [DSHB], F1.652s, 1:20), Pax7 (DSHB, Pax7c, 1:100), MHC I (DSHB, BA-D5c, 1:100), MHC IIa (DSHB, SC-71c, 1:100), MHC IIb (DSHB, BF-F3c, 1:100), laminin (Abcam, ab7463, 1:200), Ly6G (Gr-1) (BD Biosciences, BD550291, 1:50), CD68 (Bio-Rad, MCA1957, 1:50), and CD163 (Santa Cruz Biotechnology, sc-33560, 1:50). Primary antibodies used on rat muscle included MHC type I (DSHB, BA-D5c, 1:100), MHC IIa (DSHB, SC-71c, 1:100), MHC IIb (DSHB, BF-F3c, 1:100), HIS48 (Abcam, Ab33760, 1:20), CD68 (Abcam, ab31630, 1:50), and CD163 (Santa Cruz Biotechnology, sc-33560, 1:50). Antibody binding was visualized with standard Alexa Fluor secondary antibodies (Invitrogen, Thermo Fisher Scientific, 1:500 in PBS), except for Pax7, which was detected using a Tyramide SuperBoost Kit (Invitrogen, Thermo Fisher Scientific B40913). Fluorescent dyes DAPI (Invitrogen, Thermo Fisher Scientific, D21490, 2 μg/mL), wheat germ agglutinin (WGA) Alexa Fluor 647 conjugate (Invitrogen, Thermo Fisher Scientific, W32466, 5 μg/mL), WGA CF405S conjugate (Biotium, 29027, 100 μg/mL), and phalloidin (Invitrogen, Thermo Fisher Scientific ActinRed 555 ReadyProbes, R37112) were used to counterstain cell nuclei, extracellular matrix, and muscle fibers, respectively.