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6 protocols using dmrxa2 fluorescence microscope

1

Placental Vasculature Quantification

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Fluorescence micrographs were acquired using a Leica DMRXA2 fluorescence microscope equipped with a Leica DFC350FX camera. Histochemical micrographs were acquired using a Leica DMRXA2 fluorescence microscope equipped with a Leica DFC300FX camera. Images were processed using Adobe Photoshop.
Blood vessels in placenta labyrinth were counted as previously described [45] (link). Statistical analysis was conducted using unpaired t-test by GraphPad Prism 4. Results with p values less than 0.05 were considered significant.
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2

Chromosome Preparation and In Situ Hybridization

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Chromosome preparations and in situ hybridization were performed as described in D’Hont et al. (2000) with modifications. BAC clones (Table S3) from accession DH‐Pahang (D’Hont et al., 2012; http://banana‐genome.cirad.fr) were labeled by random priming with biotin‐14‐dUTP (Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA) or Alexa 488‐5‐dUTP (Life Technologies; Thermo Fisher Scientific). Chromosome preparations were incubated in RNAse A (100 ng µl–1) and pepsin (100 mg ml–1) in 0.01 m HCl and fixed with paraformaldehyde (4%). Biotinylated probes were immunodetected by Texas Red avidin DCS (Vector Laboratories, Burlingame, CA, USA) and the signal was amplified with biotinylated antiavidin D (Vector Laboratories). Fluorescence images were captured using a cooled high‐resolution black and white CCD camera (ORCA; Hamamatsu, Hamamatsu City, Japan) fitted on a DMRXA2 fluorescence microscope (Leica Microsystems, Wetzlar, Germany) and analysed using volocity (Perkin Elmer, Waltham, MA, USA).
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3

Characterization of Pluripotency and Differentiation Markers

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The ES R1 and EC F9 cells were fixed with 2% paraformaldehyde in phosphate-buffered saline, PBS, pH 7.0, within 15 min. ALP activity was detected after incubation in a solution containing 10 ml 0.02 M Tris-HCl buffer (pH 8.7), 1 mg Naphtol-AS-B1-phospate, and 5 mg fast red dye Texas Red (all from Sigma) at 37°C for 1 h.
For immunofluorescence analysis, cells fixed in 4% paraformaldehyde in PBS for 1 h were washed and permeabilized with 0.5% Triton X-100. Nonspecific reactions were blocked by 10% chicken serum (Gibco/Invitrogen). Primary rabbit anti-Oct4 and goat anti-Gata4 antibodies (Santa Cruz Biotechnology) were used at a dilution of 1 : 100. The cells were incubated in a solution of primary antibodies in PBS-Tween 20 at 4°C overnight. Secondary chicken anti-rabbit and donkey anti-goat antibodies conjugated with Alexa Fluor 594 and Alexa Fluor 488 (Molecular Probes) were diluted at 1 : 800 in a blocking buffer and applied to the cells for 4 h at room temperature. DAPI (Molecular Probes) was applied for 15 min for nuclear staining. The cells were mounted and examined under a Leica DMRXA2 fluorescence microscope (Leica Microsystems GmbH). For negative controls, the primary antibodies were omitted, and the same staining procedure was used.
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4

Characterization of NMBs on Glass Slides

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A Pro-4 four point system (Lucas Labs, Gilroy, CA, USA) was used in resistivity measurements. The system was made up of a Keithley 2400, a Pro4, and a notebook with the Pro4 V1.2.4 software installed. The system uses the dual configuration test method of ASTM standard F84-99 to compensate for errors in probe spacing and errors caused by proximity to the edge of the conducting layer. A V/I measurement was taken and recorded and the subsequent resistivity was computed.
Scanning electron microscope (SEM) micrographs were obtained using a Quanta 3D FEG Dual Beam FIB/SEM and its xT Microscope Control Software (FEI, Hillsboro, OR, USA). Fluorescence studies were performed on samples using a DM RXA2 fluorescence microscope (Leica, Wetzlar, Germany) fitted with a 100× objective lens with a 1.4 N.A. Slidebook version 4.1 was used for image acquisition from a SensiCam QE camera (Cooke, Auburn Hills, MI, USA) attached to the microscope.
Optical images were obtained from a 40 CFL inverted microscope (Axiovert, Thornwood, NY, USA) fitted with a 20× objective with a 1.0 N.A. AxioVision 4.6.1.0 was used for image acquisition from an AxioCam MRc camera attached to the microscope. Percentage area covered by the NMBs on the glass slides were determined using the ImageJ 1.46r software (Wayne Rasband, National Institutes of Health, USA).
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5

Immunofluorescence Staining of γH2AX

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The cells covered 80–90% of the glass coverslips. The cells were washed three times with PBS and fixed in 4% paraformaldehyde (Boster, Wuhan, China). Then, the cells were permeabilized with 0.1% Triton X-100 and blocked in 4% bovine serum albumin. Images were obtained using a DM-RXA2 fluorescence microscope (Leica) after incubation with γH2AX antibody (Merck Millipore, Darmstadt, Germany), a fluorescence-conjugated secondary antibody, and DAPI (4′,6-diamidino-2-phenylindole) stain (Helixgen, Guangzhou, China).
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6

Visualizing Actin and Cell Nuclei

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Following 18 and 72 hours of culture, cells were washed with PBS and fixed using a solution of 3.7% paraformaldehyde for 10 min. The fixed cells were washed again with PBS and permeabilised with 0.5% Triton X-100 for 10 min. Tetramethylrhodamine isothiocyanate conjugated phalloidin (Pha-TRITC) 0.2 μg mL -1 (Sigma, St. Louis, MO, USA) was used to visualise the actin cytoskeleton. Additionally, the top surface of the discs was coated with Fluoroshield antifade mountant containing 4',6-diamidino-2-phenylindole (DAPI) (Vector Laboratories) to stain cell nuclei. For the 18-hour time point wheat germ agglutinin-AlexaFluor488 conjugate was used at a concentration of 5 µg mL -1 to stain the cell membranes according to manufacturer's instructions. Samples were examined using a Leica DMRXA2 fluorescence microscope.
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