mTurquoise2 blue fluorescent protein fused with KDEL or Mitochondria targeting sequences were designated as cellular organelle markers of ER or mitochondria, respectively. The cells expressing each turquoise2 cultured on glass bottom plate were incubated with media containing JG compounds at 37°C for 120 min and imaged using a LSM-700 scanning confocal microscope (Zeiss). The images were merged with image J software.
Lsm 700 confocal scanning microscope
Manufactured by Zeiss
Sourced in Germany
The LSM 700 is a confocal scanning microscope manufactured by Zeiss. It is designed to provide high-resolution imaging of samples by scanning them with a focused laser beam and detecting the emitted fluorescence or reflected light. The LSM 700 is capable of producing detailed 2D and 3D images of biological, materials, and other specimens.
Automatically generated - may contain errors
Lab products found in correlation
Ab228549, by Abcam (2 mentions)
Ab5450, by Abcam (2 mentions)
Ab150129, by Abcam (2 mentions)
A 21207, by Abcam (2 mentions)
Glass bottom petri dish, by MatTek (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Superfrost plus glass slide, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Oct embedding medium, by Thermo Fisher Scientific (1 mentions)
Cryostat, by Leica (1 mentions)
Cytosoft imaging 24 well plates, by Advanced BioMatrix (1 mentions)
Duolink pla kit, by Merck Group (1 mentions)
H 1000, by Vector Laboratories (1 mentions)
F9665, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Antifade mounting medium, by Vector Laboratories (1 mentions)
Axio imager m2 microscope, by Zeiss (1 mentions)
Tunel assay kit fitc, by Abcam (1 mentions)
26 protocols using lsm 700 confocal scanning microscope
1
Organelle-Specific Fluorescent Markers
2
Immunohistochemical Labeling in Mouse Tissue
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Mice were anesthetized with ketamine/xylazine (200/14 mg/kg, i.p.), perfusion-fixed (4% PFA/0.1 M PB) and sectioned using a vibratome (30 μm sections, 1:3 serial). Sections were stored at −20°C in cryoprotectant solution consisting of the following: 0.05 M sodium phosphate buffer (PB), 30% ethylene glycol, 20% glycerol. All primary and secondary antibodies used in this study are listed in Table 1 . Upon removal from cryoprotectant solution, sections were washed in 0.1 M PB (3 × 5 min) and then Tris saline (TS), pH 7.4 (3 × 5 min). Sections were blocked in TS containing 0.3% Triton X-100 and 10% fetal horse serum (FHS) at room temperature for 45 min. Sections were then incubated in primary antibody solution (TS/0.1% Triton X-100/1% FHS) overnight at 4°C with gentle rocking. After primary antibody incubation, sections were washed in TS (3 × 10 min) before incubation for 90 min at room temperature in secondary antibody solution (TS). DAPI solution was added during the last minute of secondary antibody incubation period. Sections were mounted on Superfrost Plus glass slides (Fisher Scientific 12-550-15) and sealed with ProLong Gold antifade reagent with DAPI (Invitrogen P36935) before imaging on a Zeiss AxioImager Z1 widefield epifluorescence microscope (Figs. 3 , 5 , 7 , 8 ) or a Zeiss LSM700 scanning confocal microscope (Figs. 2 , 9 , 10 ).
3
Histochemical Analysis of Tentacle Structures
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Tentacle samples were rinsed in 0.2 µm-filtered sterile artificial seawater (SASW) (91 ), and then fixed in 4% paraformaldehyde in SASW for 30 min, rinsed with PBS + 0.1% Triton-X100 (PBST), and incubated with Alexa-Fluor-555-conjugated phalloidin (Invitrogen) diluted 1:250 and DAPI (ThermoFisher) diluted 1:10,000 in PBST overnight at 4 °C with gentle agitation. Following staining, samples were thoroughly washed in PBST, and then transitioned through a series of 15% and 30% sucrose in PBS and incubated in OCT embedding medium (Fisher Sci.) overnight at 4 °C. Samples were then snap-frozen on dry ice and sectioned on a cryostat (Leica). Sections were mounted and imaged on a Zeiss LSM-700 scanning confocal microscope.
4
Quantifying Protein Interactions via PLA
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From 25,000 to 50,000 cells were seeded on 35-mm glass-bottomed dishes (WPI). For substrate stiffness experiments, CytoSoft Imaging 24-well plates of 0.2 kPa and 64 kPa stiffness (Advanced Biomatrix) were used. The Duolink PLA Kit (Millipore Sigma) was used per manufacturer’s instructions. Specifically, the mouse minus, rabbit plus, and orange detection kit were used together. Cells were initially fixed with ice-cold 99% methanol. After the cells were treated per the PLA protocol, they were imaged within 24 h using an LSM 700 scanning confocal microscope (Carl Zeiss) with a 63×, 1.4 NA objective. Images were analyzed using Fiji through the “Analyze Particles” function or by measuring the raw integrated density of the entire cell, as indicated within the individual figures, and normalized to cell spread area as indicated in the figures.
5
Immunofluorescence Microscopy of Fixed Cells
6
Immunofluorescence Staining of Tissue Samples
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Immunofluorescence staining was performed on cryosections and whole-mount dissections. The tissues and slides were washed with blocking solution [2% donkey serum (Sigma-Aldrich, S30), 5% fetal bovine serum (FBS, Sigma-Aldrich, F9665), and 0.1–0.5% triton X-100 (Sigma-Aldrich, X100) in PBS]. Specimens were incubated with primary antibodies, anti-myosin VIIA (MYO7A, 1/200, rabbit, Proteus Biosciences, 25-6790) and anti-GFP (1/200, goat, Abcam, ab5450) overnight at 4°C. Later, slides and tissues were washed with blocking solution and incubated with secondary antibodies donkey-anti Rabbit-Alexa 594 (1/500, Invitrogen, A-21207), donkey-anti Goat-Alexa 488 (1/500, Abcam, AB150129), and DAPI solution (1/500, Abcam, AB228549) for 90 min at room temperature. Lastly, specimens were washed in PBS and mounted in Vectashield Antifade Mounting Medium (Vector laboratories, H-1000). Slides were imaged using a Zeiss LSM700 Scanning Confocal Microscope. Three-dimensional image reconstructions of Z-stacks were performed using ImageJ software.
7
Quantifying Organoid Clearing Depth
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We stained the organoids with 0.5 μg/mL DAPI (Sigma) in PBS for 24 hr and subsequently subjected them to the different tissue clearing protocols. Afterwards, a LSM 700 scanning confocal microscope (Zeiss) was used to acquire z-stacks of the stained and cleared organoids. Three XZ and three YZ cross-sections per aggregate (n = 10 aggregates per clearing method) were used to quantify the maximum depth at which the DAPI signal could still be detected at a given brightness threshold. The depth of each cross section was measured manually at n = 5 different positions for each slice and n = 10 organoids per clearing protocol using ImageJ/Fiji. The data was exported to Microsoft Excel for further processing and GraphPad Prism v7.0 for plotting and statistical analysis.
8
Imaging Cochlear and Vestibular Cells
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Cochlear and vestibular explants from Cib2 mutant and control mice were dissected at postnatal day 5 (P5) and cultured in a glass-bottom petri dish (MatTek, Ashland, MA). They were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS (Life Technologies) for 2 days at 37 °C and 5% CO2. Explants were incubated for 10 s with 3 µM FM1-43, washed three times with Hank's balanced salt solution, and imaged live using a Zeiss LSM 700 scanning confocal microscope.
9
Immunofluorescence Staining of Cochlear Tissue
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Immunofluorescence staining was performed on cryosections and whole-mount dissections. The tissues and slides were washed with blocking solution (2% donkey serum and 0.1% triton X-100 in PBS). Specimens were incubated with primary antibodies, anti-myosin VIIA (MYO7A, 1/300, rabbit, Proteus Biosciences, 25-6790) and anti-GFP (1/200, goat, Abcam, ab5450) overnight at 4°C. Later, slides and tissues were washed with blocking solution and incubated with secondary antibodies donkey-anti Rabbit-Alexa 594 (1/500, Invitrogen, A-21207), donkey-anti Goat-Alexa 488 (1/200, Abcam, AB150129), and DAPI solution (1/500, Abcam, AB228549) for 90 min at room temperature. Lastly, specimens were washed in PBS and mounted in Vectashield Antifade Mounting Medium (Vector laboratories, H-1000). Slides were imaged using a Zeiss LSM700 Scanning Confocal Microscope. Apical, middle, and basal regions were calculated by measuring the total length of each cochlear duct in the whole-mount dissections and calculating 25% (apex), 50% (middle), and 75% (basal) distance from the apical end. Three-dimensional image reconstruction of Z-stacks and PCC analyses of DAPI and GFP signals were performed using ImageJ software.
10
Visualization of Hair Cell Calcium Entry
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In the Ahmed lab, cochlear explants from Cib2 and Cib3 mutant mice were dissected at postnatal day 5 (P5) and cultured in a glass-bottom petri dish (MatTek, Ashland, MA). They were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS (Life Technologies) for 2 days at 37 °C and 5% CO2. Explants were incubated for 10 s with 3 μM FM 1–43, washed three times with Hank’s balanced salt solution, and imaged live using a Zeiss LSM 700 scanning confocal microscope.
In the Holt Lab, P60 mice were injected intraperitoneally with 5 mg/g mouse weight of FM 1–43FX fixable form as previously described (Meyers et al., 2003 (link)). Tissue was collected after ~24 h and fixed for 1 h at 25 °C in 4%PFA/1X PBS protected from light. Tissue was then transferred into 120 mM EDTA pH 7.4 for 48–72 h protected from light. Then tissue was transferred to 1XPBS, dissected, incubated in 1:1000 dilution of Phalloidin (Invitrogen), washed three times in 1XPBS and mounted under glass coverslips for microscopy. Imaging was completed using a 20× objective on a Zeiss 800 confocal microscope with laser intensity, detector gain, and post processing set equally for each sample.
In the Holt Lab, P60 mice were injected intraperitoneally with 5 mg/g mouse weight of FM 1–43FX fixable form as previously described (Meyers et al., 2003 (link)). Tissue was collected after ~24 h and fixed for 1 h at 25 °C in 4%PFA/1X PBS protected from light. Tissue was then transferred into 120 mM EDTA pH 7.4 for 48–72 h protected from light. Then tissue was transferred to 1XPBS, dissected, incubated in 1:1000 dilution of Phalloidin (Invitrogen), washed three times in 1XPBS and mounted under glass coverslips for microscopy. Imaging was completed using a 20× objective on a Zeiss 800 confocal microscope with laser intensity, detector gain, and post processing set equally for each sample.
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