Optogenetic stimulation experiments were performed on Zeiss LSM-510 (40×/1.0 NA objective) and a Nikon C1 (40×/0.8 NA objective). Neurod6Creand Ai27 (Gt(ROSA)26Sor) lines were crossed to yield mice that expressed ChR2 and tdTomato in AIIs and at least one other narrow-field AC type. AIIs were distinguished from other labelled amacrines due to their stronger tdTomato expression and their arboreal dendrites that extended through the entirety of the IPL and sometimes into the GCL (the dendrites of other labelled ACs were constrained to IPL sublaminas 1-4). ChR2 was activated with a 500 ms spot scan from a 488 nm Ar laser centered over an AC soma. Stimulation timing was verified by diverting a fraction of the laser light to a photodiode. ChR2-evoked IPSCs were recorded from GCs in whole-cell voltage clamp (Vhold = +10 mV) with a Cs-based internal solution supplemented with Alexa488 (50 uM). Photoreceptor-driven light responses to the 488 nm laser were eliminated by blocking all glutamatergic synaptic transmission in the retina (e.g. eliminating bipolar transmission) with L-AP4 (20 μM; Abcam), UBP 310 (25 μM; Abcam), NBQX/DNQX (10 μM/20 μM; Abcam) and D-AP5/R-CPP (25 μM/5 μM; Tocris Bioscience). AII-GC distance was determined by collecting a composite z-stack and measuring the linear distance between the centers of the AII (red) and GC (yellow) somas in the x-y plane.
Lsm 510
Manufactured by Nikon
The Nikon LSM 510 is a laser scanning microscope that provides high-resolution imaging capabilities. It is designed to capture detailed images of samples for research and analysis purposes.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst nuclear stain, by Thermo Fisher Scientific (2 mentions)
Cellevent caspase 3 7 detection reagent, by Thermo Fisher Scientific (2 mentions)
To pro 3 iodide, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Eclipse 80i, by Nikon (2 mentions)
Lsm710, by Nikon (2 mentions)
Lumox 24 multiwell plate, by Sarstedt (2 mentions)
Eclipse ti2, by Nikon (2 mentions)
C1 upright confocal microscope, by Adobe (2 mentions)
Fluoromount g, by Southern Biotech (2 mentions)
Volocity, by PerkinElmer (2 mentions)
L ap4, by Abcam (1 mentions)
Ubp 310, by Abcam (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Fugene hd, by Roche (1 mentions)
Epcam clone g8, by BioLegend (1 mentions)
Click it cocktail, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Dow (1 mentions)
A1 plus, by Nikon (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
16 protocols using lsm 510
1
Optogenetic Stimulation of AII Amacrine Cells
2
Immunofluorescence Analysis of Cystatin B and STAT-1 in HIV-Infected Macrophages
3
Visualizing Cytoskeletal Dynamics with RhoGTPases
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Cells were transfected with pcDNA3 containing either EGFP, EGFP-RhoAT19N, EGFP-RacAT17N or EGFP-Cdc42T17N using Fugene HD (Roche) transfection reagent according to the manufacturer's protocol. For microscopy, cells were fixed with 3.2% formaldehyde, permeabilized with 0.1% Triton X-100 and stained with rhodamine-phalloidin to visualize F-actin and SYTO-13 to visualize DNA as indicated. For immunofluorescence microscopy, we used α-GST, α-occludin and α-ZO-1 antibodies (Sigma) diluted 1∶500, followed by FITC-labeled α-rabbit antibody (Sigma) at a 1∶1000 dilution. Images were taken either on a Zeiss LSM 510 scanning confocal microscope or a Nikon Eclipse Ti fluorescence microscope and images were prepared using ImageJ and Corel Draw X5. For quantification of the F-actin phenotype, the total number of cells as well as number of cells containing stress fibers, were enumerated. Some fields contained cells displaying cortical actin, however this phenotype was observed across experiments and was independent of MAM adhesion. Thus, these cells were not counted as positive. Data shown are means ± standard deviation from twelve images (four frames from triplicate experiments, representing at least 100 cells/experimental condition).
4
Immunofluorescent Analysis of Distal Colon
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Distal colonic tissue was flushed of luminal contents using PBS and fixed for 1 hour in 2% PFA (paraformaldehyde), dehydrated in 30% sucrose overnight at 4°C, and flash-frozen in OTC (organotypic 3D culture) media. Sections were stained with antibodies specific to EPCAM (clone G8.8, catalog #118212; BioLegend), phospho-PDH (Ser 293, ref 31866; Cell Signaling), and Ki67 (clone SolA15, ref 14-5698-82; Invitrogen) overnight, and for 5 minutes for the Hoechst nuclear stain (ref H3570; Invitrogen). For EdU identification, slides were permeabilized with Triton-X-100 (Dow) for 10 minutes and stained in Click-It cocktail (ThermoFisher) for 20 minutes at room temperature. Images were taken on Zeiss LSM 510 and Nikon A1 confocal microscopes and analyzed using ImageJ software.
5
Quantitative Immunofluorescence for DNA Damage
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Immunofluorescence for γH2AX was performed using a standard protocol (details in Supplemental Material ); a minimum of 40 cells per mouse were evaluated (blinded) for each experiment. Activated Caspase 3/7 was measured with the CellEvent Caspase 3/7 detection reagent (ThermoFisher) according to the manufacturer’s instructions. Nuclei were counterstained with DAPI (Sigma-Aldrich) or ToPro3-iodide (Invitrogen) and images captured using microscopy (Zeiss LSM 510, Nikon A1R, or Nikon Eclipse 80i); a minimum of 110 cells per mouse were evaluated (blinded) for each experiment.
6
Perfusion-Fixed Brain Sectioning and Immunostaining
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Mice of the appropriate age were anesthetized and perfused transcardially with 4% paraformaldehyde/PBS as previously described [10 (link), 12 (link), 14 (link)]. Brains were dissected, postfixed, and mounted in agarose prior to vibratome sectioning. Overall, 35–100 μm free-floating vibratome sections of brains were collected in PBS, blocked with 5% normal serum in PBS with 0.5% Triton X-100 and incubated with primary antibodies in blocking solution overnight at 4 °C. P14 vibratome sections were blocked with 5% normal serum in PBS with 0.1% Triton X-100 and 2% DMSO, and incubated with primary antibodies for 3 days. After 1–3 h incubation with Alexa Fluor-conjugated secondary antibodies (Invitrogen or Jackson Immunoresearch) followed by DAPI staining (0.1 μg/ml, Life Technologies), the stained slices were imaged using a confocal laser-scanning microscope (Zeiss LSM510, LSM710 or Nikon A1 plus).
7
Imaging of AT1-like and D2.0R Cell Dynamics
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2x104 AT1-like cells/well were plated onto Lumox 24-multiwell plate (Sarstedt, 94.699.00.14) in MLNL or MHNH medium as indicated. The following day 2000 D2.0R cells were plated in the same media. 3-4 hours after plating the indicated inhibitors were added and imaging for 48 hours using either a LSM510 or Nikon Eclipse Ti2 was commenced two hours later. The movies were analyzed manually to record the number of cells at the beginning, at the end, the number of mitoses, and the number of cell death events.
8
Larval Midgut Ultrastructure Analysis
9
Quantitative Immunofluorescence for DNA Damage
10
Immunostaining of Drosophila Wing Discs
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For each sample, 10–20 larvae were dissected on ice-cold phosphate-buffered saline (PBS) and fixed in 4% formaldehyde in PBS overnight at 4°C. After fixation, the tissue was washed four times (15 min per wash) with 0.3% Triton X-100 in PBS (PBT), then blocked for 30 min at room temperature in 2% heat-inactivated normal donkey serum in PBT. After blocking, the tissue was incubated in a primary antibody solution diluted with 2% heat-inactivated normal donkey serum in PBT overnight at 4°C. We used the guinea pig anti-Senseless (Nolo et al., 2000 (link), 1:1000) and mouse anti-Achaete (Developmental Studies Hybridoma Bank, contributor J. Skeath, supernatant, 1:10) primary antibodies. To compare signal across tissues, we stained for both antigens simultaneously. The washing and blocking procedure was repeated after primary antibody incubation, and then the tissue was incubated in a secondary antibody (1:200 each of anti-guinea pig [Alexa Fluor 546] and anti-mouse [Alexa Fluor 488]) overnight at 4°C. The tissues were washed with PBT and rinsed with PBS, and then the wing imaginal discs were mounted on poly-l -lysine-coated coverslips using Fluoromount-G (SouthernBiotech). Tissues were imaged using either a Leica LSM 510 or a Nikon C1 upright confocal microscope and processed using ImageJ (version 2.0) and Adobe Photoshop CC 2017.
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