Fluorescence imaging was performed as previously described (Kowalski, Dahlberg & Juo, 2011 (link)). Briefly, nuIs24 or nuIs24; dmd-10(gk1131) young adults were immobilized using 30 mg/ml 2,3-butanedione monoxamine (Sigma-Aldrich, St. Louis, MO, USA) and the anterior ventral nerve cord immediately posterior to RIG was imaged (Juo et al., 2007 (link)). A total of 1 μm (total depth) Z-series stacks were collected using a Carl Zeiss Axiovert M1 microscope with a 100X Plan Apochromat (1.4 numerical aperture) objective equipped a GFP filter cube. Images were collected with an Orca-ER CCD camera (Hamamatsu) and MetaMorph (version 7.1) software (Molecular Devices). Maximum intensity projections of Z-series stacks were used for quantitative analyses of fluorescent puncta. Exposure settings and gain were adjusted to fill the 12-bit dynamic range without saturation and were identical for all images taken. Line scans of ventral cord puncta were generated using Meta-Morph (version 6.0) and were analyzed using custom written software (Burbea et al., 2002 (link)) with Igor Pro (version 6 Wavemetrics). Statistical analyses were performed using Student’s t test.
Axiovert m1 microscope
Manufactured by Zeiss
The Axiovert M1 is a compact and versatile inverted microscope designed for a wide range of applications in life sciences and materials research. It features a high-quality optical system and a robust mechanical construction to ensure reliable performance and consistent results.
Automatically generated - may contain errors
Lab products found in correlation
2 3 butanedione monoxamine, by Merck Group (3 mentions)
Orca er ccd camera, by Hamamatsu Photonics (2 mentions)
Plan apochromat, by Zeiss (2 mentions)
Meta morph, by Wavemetrics (2 mentions)
Metamorph, by Molecular Devices (2 mentions)
Igor pro, by Wavemetrics (1 mentions)
Metamorph version 7.1, by Molecular Devices (1 mentions)
Fluosphere beads, by Thermo Fisher Scientific (1 mentions)
Orca er charge coupled device camera, by Hamamatsu Photonics (1 mentions)
Plan aprochromat, by Zeiss (1 mentions)
Aprochromat objective, by Zeiss (1 mentions)
Orca er, by Hamamatsu Photonics (1 mentions)
Tetramisole, by Merck Group (1 mentions)
Yfp filter, by Zeiss (1 mentions)
5 protocols using axiovert m1 microscope
1
Fluorescence Imaging of Ventral Nerve Cord
2
Fluorescence Imaging of Ventral Nerve Cord
3
Quantitative Analysis of GFP Reporter Fluorescence
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All GFP reporter imaging experiments were performed with a Carl Zeiss Axiovert M1 microscope with a 100x Plan Aprochromat objective (1.4 numerical aperture) with GFP and RFP filter cubes. Images were acquired with an Orca-ER charge-coupled device camera (Hamamatsu), using MetaMorph, version 7.1 software (Molecular Devices). All L4 animals were immobilized with 30 mg/ml 2,3-butanedione monoxamine (Sigma-Aldrich, St. Louis, MO) for 6–8 minutes before imaging. To quantitate GFP fluorescence, maximum intensity projections from Z-series stacks of 1 μm depth were taken from the PVC nucleus using MetaMorph software. Exposure settings were constant for each reporter. A region of interest was drawn around the nucleus and maximum pixel intensity was used for quantification. At least 20 animals were measured for each genotype and statistics were performed by Student’s t test (for two genotypes) or ANOVA with Tukey-Kramer post hoc correction (greater than two genotypes). Control genotypes were always assayed on the same day to normalize for daily fluctuations in fluorescence.
4
Quantitative Imaging Analysis of C. elegans Neurons
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Imaging was performed with a Carl Zeiss Axiovert M1 microscope with 100x Plan Aprochromat objective. Images were taken with a Hamamatsu Orca-ER charge-coupled device and processed with MetaMorph, version 7.1 software (Molecular Devices). L4 hermaphrodites were used for all imaging. Animals were paralyzed with 30 mg/mL 2, 3-butanedione monoxamine (BDM, Sigma-Aldrich) for 5 minutes before imaging. For quantitative VNC imaging, maximum intensity Z-series stacks, 1 μm total thickness, were taken from the anterior VNC. FluoSphere fluorescent beads were used to normalize image fluorescence intensity. Line scans were made using MetaMorph (v6.0) and analyzed using IgorPro (v5) and custom-written software [52 (link)]. Constant exposure settings were used across genotypes and across all imaging days. Cell counts of GLR-1-expressing neurons marked with Pglr-1::NLS-GFP-LacZ (pzIs29), NMR-1-expressing neurons marked with Pnmr-1::GFP (akIs3), ASH neurons stained with DiI and ASH neurons marked with ChR2::YFP (ljIs114) were done manually by focusing through the worm head using a 63X objective.
5
Quantifying polyQ40::YFP Aggregation in C. elegans
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The following strains were used in this study: N2 (Bristol), rmIs133 [unc-54p::Q40::YFP] (polyQ40::YFP expressed under a muscle-specific promoter) 43 (link) , cdc-48.1(tm544), ubxn-1(tm2759) and ubxn-4(ok3343). All strains were maintained at 20 o C as described previously 53 .
Representative fluorescent images of polyQ40::YFP in L4 larval stage C. elegans were acquired using a Carl Zeiss Axiovert M1 microscope equipped with a 5X objective and a YFP filter (Zeiss). Images were collected with an Orca-ER charged coupled device (CCD) camera (Hamamatsu) and Metamorph (version 7.1) software. Exposure settings and gain were adjusted to fill the 12-bit dynamic range without saturation and were identical for all images. Larval-stage 4 (L4) animals were immobilized in a droplet of M9 containing 2.3mM levamisole (Tetramisole, Sigma) and placed on a 2% agarose pad containing 3.4 mM levamisole. To quantify fluorescent puncta, worms were precisely age-matched at the L4.4 vulva development stage based on vulva morphologyusing a 100X objective. Then the number of polyQ40::YFP aggregates were visually counted on the microscope using the 5X objective and recorded. All genotypes were blinded prior to the experiment. At least 3-5 animals were measured for each genotype across 3 independent experiments and statistical analyses were performed by ANOVA followed by Dunnett's multiple comparisons test.
Representative fluorescent images of polyQ40::YFP in L4 larval stage C. elegans were acquired using a Carl Zeiss Axiovert M1 microscope equipped with a 5X objective and a YFP filter (Zeiss). Images were collected with an Orca-ER charged coupled device (CCD) camera (Hamamatsu) and Metamorph (version 7.1) software. Exposure settings and gain were adjusted to fill the 12-bit dynamic range without saturation and were identical for all images. Larval-stage 4 (L4) animals were immobilized in a droplet of M9 containing 2.3mM levamisole (Tetramisole, Sigma) and placed on a 2% agarose pad containing 3.4 mM levamisole. To quantify fluorescent puncta, worms were precisely age-matched at the L4.4 vulva development stage based on vulva morphologyusing a 100X objective. Then the number of polyQ40::YFP aggregates were visually counted on the microscope using the 5X objective and recorded. All genotypes were blinded prior to the experiment. At least 3-5 animals were measured for each genotype across 3 independent experiments and statistical analyses were performed by ANOVA followed by Dunnett's multiple comparisons test.
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