To assess the distribution of NP1 protein in different brain regions and sub-regions (e.g. hippocampal CA1, CA3 and dentate gyrus) representative coronal brain sections (20 μm) were subjected to immune-staining and analyzed by fluorescence microscopy as described previously (Hossain et al., 2004 (link)). Mouse pups were sacrificed at the indicated time-points after HI by perfusion fixation with ice-cold 4% paraformaldehyde in 1X PBS and cryoprotected sequentially in 15% and 30% sucrose solutions for overnight followed by freezing on dry ice. Representative coronal brain sections (20 μm) from control and HI animals were immunostained as described previously (Al Rahim et al., 2013 (link); Hossain et al., 2004 (link)). Mouse monoclonal anti-NP1 (1:200) (BD Transduction Laboratories, Temecula, CA, USA), GFAP (1:1000), COXIV (1:250) and cleaved caspase-3 (1:200; Cell Signaling, Beverly, MA, USA) was used as the primary antibody, while donkey anti-mouse, anti-rabbit Alexa fluor 488 (green) and 568 (red) are the secondary antibodies used (Invitrogen, Carlsbad, CA, USA). Immunofluorescence was visualized using an inverted fluorescence microscope (Olympus IX51 fitted with DP2-DSW-V3.2 application software) at 10 X and ZEISS Axioimager M2 (AxioVision SE64 Rel. 4.8.1 application software) at 100 X magnification.
M2 axioimager
Manufactured by Zeiss
Sourced in United States
The M2 AxioImager is a microscopy platform designed for advanced imaging applications. It features a robust and modular design that accommodates a range of objectives and accessories to support various research and analysis needs.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Cox 4, by Cell Signaling Technology (1 mentions)
Glial fibrillary acidic protein (gfap), by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti glur1, by Merck Group (1 mentions)
Zen 2012, by Zeiss (1 mentions)
Dfc480 camera, by Leica (1 mentions)
Stemi 2000 c, by Zeiss (1 mentions)
As lmd laser dissection microscope, by Leica (1 mentions)
Axiocam erc5s camera, by Zeiss (1 mentions)
Calcofluor white, by Merck Group (1 mentions)
Axio imager, by Zeiss (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Toluidine blue, by Merck Group (1 mentions)
Ultracut r microtome, by Leica (1 mentions)
Levamisole, by Thermo Fisher Scientific (1 mentions)
Plan apochromat, by Zeiss (1 mentions)
Lsm 710 confocal laser scanning microscope, by Zeiss (1 mentions)
1 1 dioctadecyl 3 3 3 3 tetramethylindocarbocyanine perchlorate, by Thermo Fisher Scientific (1 mentions)
Zen 2, by Zeiss (1 mentions)
13 protocols using m2 axioimager
1
Immunohistochemical Analysis of NP1 Protein Distribution
2
Live Visualization of NP1 and GluR1 in Cortical Neurons
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Live double-immunofluorescence staining of primary cortical cultures (DIV 12) with NP1 was done as described previously [7 (link),22 (link)]. Briefly, cortical neurons, grown on coverslips, following exposure to OGD (4 h) were live labeled with both anti-NP1 (1:100; BD Transduction Laboratories) and anti-GluR1 (1:100; Millipore) by adding directly to the medium and further incubated for 45 min at 37°C. Neurons were then fixed with 3.7% formaldehyde, and permeabilized cells were stained with anti-mouse Alexa Fluor 568 (red, NP1) and anti-rabbit Alexa fluor 488 (green, GluR1)-conjugated secondary antibodies (Invitrogen). Slides were coverslipped with prolong mounting medium containing DAPI (blue) (Molecular Probes, Eugene, OR, USA) to stain nuclei. Immunofluorescence was visualized using an inverted fluorescence microscope (Olympus IX51fitted with DP2-DSW-V3.2 application software) at 10 × and ZEISS Axioimager M2 (AxioVision SE64 Rel.4.8.1 application software) at 100 × magnification.
3
Confocal Microscopy and Tubule Analysis
4
Quantifying ETS-4 Localization in C. elegans
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To determine the localization of ETS-4, we constructed a plasmid (pets-4::ets-4::gfp::unc-54) containing the ets-4 promoter (2149bp), coding region (1389bp), gfp sequence, and unc-54 3’UTR, which we co-injected with a rol-6 dominant mutant plasmid (pRF4) to generate transgenic strains. Using a Zeiss fluorescence microscope (Axio Imager.M2) and Axiovision 4.8 software, we acquired images in the green and transmitted light channels (with differential interference contrast) using a 10x objective. We defined the nucleus and intestine cell regions of worms using the ROI tool in ImageJ (NIH; http://rsb.info.nih.gov/ij/ ) from DIC and GFP images. The ratio of nuclear intensity was calculated as the intensity of the nucleus divided by the intensity of the intestine cell. The ratio of all strains injected with the ets-4:gfp plasmid was calculated relative to the N2 worms that did not receive the plasmid. We quantified 2–3 nuclei in each of 3–5 worms with higher ETS-4 expression due to inconsistent cell-to-cell localization. Prism 7 was used for statistical analysis.
5
Time-Lapse Imaging of Nematode Seam Cells
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Nematodes were mounted on a 4% agarose pad on microscope slides kept at room temperature in a dark humidity chamber when not imaging. Slides were rehydrated as needed. Images were taken every hour for 2 days, using an upright compound microscope with a mechanized stage (Zeiss M2 AxioImager and Zen software). Seam cells were identified based on their location and morphology [10 (link)].
6
Immunolabeling of Heteroxylan in P. ovata
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The protocol from Phan et al. (2016) (link) was adapted for examination of HX in P. ovata mucilage. In brief, seeds were initially imbibed in 1X PBS for 30 min at room temperature, followed by a 60 min incubation in a 10-fold diluted primary LM11 (McCartney et al., 2005 (link)) or CCRC-M110 antibody with gentle agitation. The monoclonal CCRC-M110 antibody raised against Phormium tenax xylan was shown to bind P. ovata HX following the assay described in Pattathil et al. (2010) (link). Samples were washed in 1X PBS (5 × 1 min) and subsequently incubated for 60 min in a 100-fold dilution of goat anti-rat IgM conjugated with DyLite 550 (Thermo Fisher, USA) with mild agitation. Samples were again washed with 1X PBS (5 × 1 min). Whole seeds were individually mounted in 1X PBS on single cavity microscopy slides and images taken using a Zeiss M2 AxioImager with Zeiss filter set 43 (excitation BP 545/25, beam splitter FT 570, emission BP 605/70) and an AxioCam Mrm black and white camera. Images were processed using ZEN 2012 software (Zeiss, Germany).
7
Seed Coat Developmental Imaging Techniques
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Ruthenium red staining followed the protocol from Arsovski et al. (2009) with modifications excluding pre-hydration and imbibition for 10 min. Imaging was conducted using a Zeiss Stemi 2000-C dissecting microscope with an attached AxioCam ERc 5s camera. For calcofluor white staining, mature seeds were imbibed in a solution of 0.002% (w/v) calcofluor white (Sigma-Aldrich, F3543) with 0.01% Triton X-100 (Sigma-Aldrich, T8532) in 100 ml Tris (pH 8.0) for 10 min and imaged using a Leica AS LMD laser dissection microscope with an attached DFC 480 camera. To confirm developmental stages, seeds were cleared as per Tucker et al. (2012) (link) using Hoyer’s light solution (Anderson, 1954 (link)) and observed using differential interference contrast (DIC) and Nomarski optics on a Zeiss M2 Axio imager. To observe anatomical details of seed coat development, staged seed samples were fixed in 0.25% glutaraldehyde, 4% paraformaldehyde and 4% sucrose in phosphate-buffered saline (PBS; pH 7.2), embedded in LR-White resin and sectioned to 1.0 μm before staining in 0.01% toluidine blue in 0.1% sodium tetraborate as per Aditya et al. (2015) (link).
8
Visualizing Nematode Cytoskeleton and Nuclei
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Nematodes were fixed in 4% paraformaldehyde (Electron Microscopy Science) overnight at 4°C and washed three times with water. Nematodes were placed on a 4% agar pad with phalloidin (10 unit/ml; Thermo Fisher Scientific) and DAPI (0.2–0.5 μg/ml; Thermo Fisher Scientific) or Hoechst (0.2 mM; Thermo Fisher Scientific) to stain nuclei. An Andor system micropoint laser attached to a Zeiss Axioimager with 63x objective was used to create openings in the cuticle at regular intervals along the length of the nematode to increase the penetration of dyes. Following opening of the cuticle, nematodes were incubated in the stain overnight. Successful penetration of phalloidin in sedentary nematodes was confirmed by observing fluorescence in esophageal muscle. At least ten animals were examined for each developmental time point. All light microscopy images were captured with Zen software on a Zeiss M2 AxioImager with DIC and fluorescence optics. Images were examined in FIJI and multiple images were combined using the ImageJ stitching plugin.
9
Agave Leaf Tissue Sectioning and Staining
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Embedded Agave leaf tissue was sectioned at 1 μm using a diamond knife on a Leica Ultracut R microtome. Sections were collected and dried onto poly-L-Lysine-coated microscope slides and stained with either toluidine blue (Sigma-Aldrich, United States) or methylene blue/basic fuchsin (ProSciTech Pty Ltd, Australia). Sections were viewed using a Leica light microscope (Version 4.3) and images captured with a Zeiss M2 Axio Imager fitted with an MRm Rev. 3 AxioCam.
10
Imaging mechanosensory and motor neurons
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The ALM mechanosensory neuron and the GABAergic motor neurons were imaged by mounting immobilized young adult animals in 2.5 mM levamisole (Fisher Scientific #AC187870100) on glass slides with 2% agarose pads and imaged using a 40× and 63× objective on a Zeiss M2 AxioImager.
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