Axiovert 200m

Western blot analyses, immunostaining and immunoprecipitation were performed with the following antibodies: mouse cMLCK^{6 (link)}, human cMLCK^{7 (link)}, phospho-MLC2v (gift from Dr. N. Epstein, NIH)^{45 (link)}, MLC2 (ALX-BC-1150-S-L005 Enzo Life Science), α-actinin2 (mouse monoclonal A7811 Sigma, rabbit polyclonal ab68167 Abcam), myomesin (mouse monoclonal, mMAC myomesin B4, Developmental Studies Hybridoma Bank), HA (mouse monoclonal 2367 Cell Signaling, rabbit monoclonal 3724 Cell Signaling); HA-agarose (26182 Pierce); HA-POD (12013819001 Sigma), Myc (mouse monoclonal 2276 Cell Signaling, rabbit monoclonal 2278 Cell Signaling); biotinylated Myc (B7554 Sigma); Myc-agarose (20168 Pierce); Myc-POD (11814150001 Sigma), and GAPDH (MAB374 MilliporeSigma).
Fluorescent staining for the heart sections was imaged using either Leica TCS-SP2 Laser Scanning Confocal Fluorescent Microscope System or an epifluorescent ZEISS Axiovert200M. Fluorescent staining of cardiomyocytes infected with different cMLCK mutants was performed side by side, followed by imaging with the same exposure time below the level of saturation using a ZEISS Axiovert200M. Cell size was measured using fluorescent images of neonatal cardiomyocytes or HA-positive adult cardiomyocytes using ImageJ. Mouse hearts were stained with beta-galactosidase substrate, X-gal, and were photographed as described^{46 (link)}.

KV fluid flow and cilia motility analyses were performed as described previously (Yuan et al., 2012 (link)). Fluorescent red beads (0.5 μm; Invitrogen) were microinjected into the KV and fluorescent imaging was performed on an inverted microscope (Axiovert 200M, Zeiss, Oberkochen, Germany) with a 40× objective and a CCD camera (Axiocam MRm, Zeiss) at five frames per second (fps). Fluorescent bead tracking was performed in ImageJ (NIH, Bethesda, MD). For imaging KV cilia, differential interference contrast (DIC) imaging was performed on an inverted microscope (Axiovert 200M, Zeiss) with a 63 × water objective and a high-speed camera (Phantom Miro, Vision Research, Wayne, NJ) at 1000 fps. Cilia beating dynamics were further analyzed and quantified by generating kymographs of individual motile cilia from a reconstructed time series in ImageJ (NIH).

In all the experiments, except wherever indicated, the cells were transfected, fixed, stained, mounted and directly visualized under Zeiss Axiovert 200M microscope with a 63x objective (Plan Apochromat, 1.4 NA, oil) and the cells that showed detectable fluorescent signal were scored positive.
Under conditions wherein co-transfection of GFP-Ran with mCherry-α-tubulin or HA-GAPDH was performed, for quantitation of Ran transfer, we have chosen isolated fields, where a single cell was doubly transfected and the surrounding area did not have any doubly transfected cells. This was to avoid the possibility that the transfer would have occurred from another neighbouring Ran expressing cell.
For live cell counting, COS-7 cells were transfected with indicated constructs and presence of epifluorescence was monitored by direct visualization using an inverted microscope (Axiovert 200M; Carl Zeiss, Inc.) without fixing.
For quantitation, 15–30 random fields from three independent experiments were included. Data are expressed as mean ± SD. Statistical analysis has been performed using the Student’s t-test. P-values <0.05 were considered statistically significant.

The blood of the patients was collected and sent to the laboratory within 48 h for CTC extraction. Target cells were extracted from a fresh blood sample using a negative selection method. We then sent the negatively selected CTCs (NS-CTCs) to the On-Chip Sort machine (On-Chip Biotechnologies, Tokyo, Japan) and isolated the positively-selected CTCs (PS-CTCs). We used the On-Chip Flow Ver1.8.12 software for all the cell analyses. PS-CTCs are defined as cells expressing both EpCAM and Hoechst. The details of the CTC isolation protocols are detailed in the Supplementary Materials and methods, including thyroid transcription factor-1 (TTF-1) and EpCAM expression.
To determine the purity of the PS-CTCs, H1975 cells (#CRL-5908, ATCC, VA, USA) were pre-stained with CellTrace™ Calcein Red-Orange (C34851, Thermo Scientific, MA, USA) at room temperature (RT) for 30 min. After washing twice, the cells were serially diluted and counted for the subsequent spiked tests. The recovered positively selected cells were estimated under a phase-contrast fluorescence inverted microscope (Axiovert 200 M, Carl Zeiss, Jena Deutschland). The purity was defined as the target/(target cell + non-target cell) ratio. Representative IF images of TTF-1-expressing CTCs were obtained using a phase-contrast fluorescence inverted microscope (Axiovert 200 M, Carl Zeiss, Jena Deutschland, Supplementary Figure S1).