Epididymal and epithelial markers reported previously in the literature, including cysteine-rich secretory protein 1 (CRISP1), clusterin, AR, cystic fibrosis transmembrane conductance regulator (CFTR), and cytokeratin 8 (CK8) (22 (link), 28 (link)–30 (link)) were examined in this study. Primary adult HEEs were grown to confluence on 12-mm circular glass coverslips and fixed with 3% paraformaldehyde for 15 minutes. The cells were then permeabilized with 0.1% Triton X-100 for 15 minutes, followed by blocking in 1% bovine serum albumin for 30 minutes before immunocytochemistry. Antibodies used were to CRISP1 (1:100; Sigma HPA028445), clusterin (1:100; Santa Cruz Biotechnology SC166907), cytokeratin 8 (1:400; Thermo Fisher Scientific PA532469), AR (1:300; Santa Cruz SC816), and CFTR (1:300; Cystic Fibrosis Foundation #570). Alexa Fluor 488–conjugated antirabbit IgG or Alexa Fluor 549 conjugated antimouse IgG (Jackson Immunoresearch) was used as the secondary antibody. Cells were counterstained with 6-diamino-2-phenylindole (Life Technologies) and mounted with the use of Fluorsave (Calbiochem). Samples were then analyzed with the use of a Leica DMR microscope or a Zeiss 510 Meta confocal laser scanning microscope.
Dmr microscope
Manufactured by Zeiss
The DMR microscope is a high-performance optical microscope designed for advanced imaging and analysis. It features a modular design that allows for customization and integration with a variety of accessories. The core function of the DMR microscope is to provide users with exceptional image quality and resolution for a wide range of applications.
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Lab products found in correlation
Axiocam, by Zeiss (3 mentions)
Lsm 710, by Zeiss (2 mentions)
510 meta laser scanning confocal microscope, by Zeiss (1 mentions)
Alexa fluor 488 conjugated anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Fortessa flow cytometer, by BD (1 mentions)
Axiocam hr camera, by Zeiss (1 mentions)
Macs magnetic cell separation system, by Miltenyi Biotec (1 mentions)
Sybr green, by Merck Group (1 mentions)
Dm6000b, by Zeiss (1 mentions)
Ultracut e, by Leica (1 mentions)
Vectashield with dapi, by Vector Laboratories (1 mentions)
Axiocam hrc, by Zeiss (1 mentions)
Mouse monoclonal anti alpha tubulin, by Merck Group (1 mentions)
Rabbit monoclonal anti myc, by Cell Signaling Technology (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Las af lite software, by Leica (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
Zen 2010, by Zeiss (1 mentions)
Stereo discovery v12, by Zeiss (1 mentions)
8 protocols using dmr microscope
1
Immunocytochemical Analysis of HEE Markers
2
In Situ Staining of Auxin Response
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Whole seedlings were fixed with acetone (90%) for 20min at room temperature after exposure with IAA at the indicated concentrations for 24 h. Then, the seedlings were infiltrated with staining buffer for 10 min on ice. Staining buffer consisted of 100mM sodium phosphate at pH 7.2, 10 mM EDTA, 0.2% triton X-100, 1 mM Ferricyanide, 1 mM Ferrocyanide and H2O (96%). This was followed by a second infiltration of 20 min on ice with 2mM X-GlcA cyclohexyl ammonium salt added to fresh staining buffer. Samples were then transferred to 37°C for 24 hours. Samples were then washed with increasing ethanol concentrations of 25, 50, 75 and 95% for 20min each. Samples were stored at 4°C in ethanol until imaging. Images were taken with a Leica DMR microscope equipped with a Zeiss Axiocam.
3
Quantifying Plasmodium Invasion of Erythrocytes and Reticulocytes
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Schizont stage parasites were magnetically purified using the Magnetic Cell Separation (MACS) system (Miltenyi Biotec)31 (link) and added to wells of a 96-well plate containing either erythrocytes or leukofiltered BEL-A-derived reticulocytes in culture medium. Each well contained 5 × 105 cells, with cell numbers counted using a hemocytometer, in a final volume of 200 µl. Heparin (100 mU/µl final) was used to inhibit invasion in negative controls. After ~ 16 h, invasion was quantified using cell counting and flow cytometry. For cell counting, 1.5 × 105cells were applied to a slide using a cytocentrifuge. Slides were immersed in 100% methanol fixative (15 min), Giemsa stain (10 min), and water (3 min), and imaged using a Leica DMR microscope fitted with a Zeiss AxioCam HR camera. For quantification of invasion at least 1000 cells were counted per cytospin.
For flow cytometry, cells were washed in PBSAG, stained with SYBR Green (1:1000 in PBS; Sigma-Aldrich) for 20 min at room temperature in the dark, and washed three times in PBSAG. In total, 1 × 105 cells from each well were acquired using the fluorescein isothiocyanate channel of a BD Fortessa flow cytometer.
For flow cytometry, cells were washed in PBSAG, stained with SYBR Green (1:1000 in PBS; Sigma-Aldrich) for 20 min at room temperature in the dark, and washed three times in PBSAG. In total, 1 × 105 cells from each well were acquired using the fluorescein isothiocyanate channel of a BD Fortessa flow cytometer.
4
Seed Clearing and Imaging
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Siliques were fixed o/n in fixative (Ethanol:Acetic Acid 9:1 v/v) at room temperature. The following day, the fixative was replaced with 70% ethanol. Seeds were isolated from the valves and mounted in Hoyer’s solution (Chloral Hydrate:Water:Glycerol 10:2,5:1 w/v/w) and left to clear overnight. Small seeds required only a few hours of clearing. Images were taken with a Leica DM6000B or Zeiss DMR microscope, both equipped with differential interference contrast (DIC) filters and ANDOR 5.5 Neo sCMOS cameras.
5
Ultrastructural Analysis of Plant Tissues
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Roots inoculated from nurse plants were fixed for 2 h at room temperature in 4% (v/v) glutaraldehyde and postfixed with 1% (w/v) OsO4 at 4°C overnight. Further processing of the samples and embedding in Spurr's resin was carried out as previously described (Spurr, 1969 ). For immunocytochemical analysis, tissues were embedded in Lowicryl K4 (Sigma‐Aldrich) as described (Altman et al., 1984 ) (Methods S1 ). Semithin sections (1 µm) were stained with 1 % (w/v) toluidine blue in 1% (w/v) borax (Na2B4O7). Images were acquired in the bright field mode on a Leica DMR microscope equipped with an Axiocam (Zeiss). For transmission electron microscopy (TEM) analysis, ultrathin sections (70 nm) were prepared on a Reichert‐Jung Ultracut E (Leica Microsystems). Contrasting was performed with 2% (w/v) uranyl acetate (UO2(CH3COO)2) and lead citrate solution prepared according to Reynolds (1963 ). Images were acquired on a Philips Biotwin CM100 (FEI Inc., Hillsboro, OR, USA).
6
Immunofluorescence Microscopy of Parasite Samples
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Samples were fixed at different time points in fresh 4% paraformaldehyde (PFA). The suspension was allowed to adhere onto poly-l -lysine coated slides overnight at 4°C. The slides were washed once with PBS and immunocytochemistry performed per manufacturer's instructions for rabbit monoclonal anti-myc 1:200 (Cell Signaling). Mouse monoclonal anti-alpha tubulin 1:500 (Sigma) was used simultaneously or independently using the same protocol. Secondary antibodies Alexa 488-conjugated anti-rabbit IgG and Alexa 568 conjugated anti-mouse IgG for fluorescence detection were used at 1:1000 (Molecular probes). The slides were mounted in Vectashield with DAPI (Vector Labs). Parasites were visualized on a Leica SP5 confocal microscope and acquired and analysed with the LAS AF Lite software (Leica). For quantification, samples were visualized on a Leica DMR microscope and imaged with the Zeiss AxioCam HRC and Axiovision software respectively. Cell nuclei diameters were measured using ImageJ and statistical analysis performed using Student's t-test.
7
Microscopy Imaging of Cell Cultures
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Cell cultures were documented with a Nikon Eclipse TS 100 microscope, a Leica DMR microscope or a Zeiss SteREO Discovery V12 stereomicroscope. Confocal images of immunolabelled sections or cells were taken using the confocal laser scanning microscope system LSM710 (Zeiss). Images were captured using Axio Vission Rel. 4.8 and Zen 2010 (Zeiss).
8
Callose and Lignin Detection in Plant Roots
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Wild type and vpy mutants were inoculated with nurse plants for four weeks. For callose detection, roots were fixed overnight with 4% (v/v) paraformaldehyde (Fig. 6), or with 1:3 acetic acid:ethanol (Table S1), washed 5x with water, followed by staining with 0.01% (w/v) aniline blue in 150 mM KH2PO4 pH=9.5 for 48h. Epifluorescence images were acquired with a Leica DMR microscope equipped with an Axiocam (Zeiss).
Lignin was stained with phloroglucinol solution (100 ml 95% EtOH, 16 ml HCl conc., 0.1g phloroglucinol) for 30 min and directly analyzed.
Lignin was stained with phloroglucinol solution (100 ml 95% EtOH, 16 ml HCl conc., 0.1g phloroglucinol) for 30 min and directly analyzed.
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