Cells grown in liquid media as indicated in each experiment were layered on a 1% agarose pad in LB and imaged using phase-contrast with a 100× oil immersion lens objective and a Nikon Eclipse Ti-E microscope equipped with a Nikon DS-QI1 cooled digital camera. See the supplemental material for details about cell measurements.
Ds qi1
Manufactured by Nikon
Sourced in Japan
The DS-Qi1 is a high-quality digital camera designed for microscope imaging applications. It features a 1.5-megapixel CMOS sensor, a USB 2.0 interface, and supports various image capture and analysis software.
Automatically generated - may contain errors
Lab products found in correlation
Normal goat serum (ngs), by Vector Laboratories (2 mentions)
Alexa fluor 488 green goat anti mouse, by Thermo Fisher Scientific (2 mentions)
Eclipse ti e microscope, by Nikon (1 mentions)
Eclipse ti u, by Nikon (1 mentions)
Fura 2 am, by Bio-Techne (1 mentions)
Te2000 inverted microscope, by Nikon (1 mentions)
Fura 2 am, by Warner Instruments (1 mentions)
Dic n2 plan apo, by Nikon (1 mentions)
Glycerol bp229 1, by Thermo Fisher Scientific (1 mentions)
Eclipse 90i microscope, by Nikon (1 mentions)
Mouse anti human osteocalcin, by Santa Cruz Biotechnology (1 mentions)
Multiclamp 700 amplifier, by Molecular Devices (1 mentions)
Eclipse fn1 microscope, by Nikon (1 mentions)
Pclamp 10, by Molecular Devices (1 mentions)
9 protocols using ds qi1
1
Microscopic Imaging of Cells
2
Fluorescent Droplet Imaging and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The droplets were stained with SYBR Green dye I (Life Tech, Rockland, ME, USA) by adding 2 μL of 100× dye solution to 2 μL of droplet stabilization oil. The dye passively migrates between the droplets and stains dsDNA. Fluorescence images were recorded with a 1.5 megapixel digital camera (Ds-Qi1, Nikon, Tokyo, Japan) on an inverted microscope (Nikon Ti-U Eclipse). Fluorescence excitation was set at 470 ± 20 nm (with a 100-ms exposure) and emitted light collected at 525 ± 25 nm. Recorded images were processed with open-source software Fiji [28 (link)] to count the total number of droplets, the number of fluorescent droplets, mean fluorescence intensity values and the coefficient of variation.
3
Intracellular Calcium Imaging in Rat DRGs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Intracellular calcium imaging experiments were performed as previously described (Dembla et al., 2017 (link)). Briefly, cultured rat DRGs were incubated at room temperature with 5 μM Fura 2 AM: (Tocris, Wiesbaden-Nordenstadt, Germany) (1 mM stock in DMSO +0.02% pluronic F-127) for 30 min in growth medium. Following the application of Fura 2 AM, cover slips were transferred to a closed recording chamber (Warner Instruments, Hamden, CT, USA) and continuously perfused with the extracellular solution for 5 min. Fluorescence was monitored every 2 s and images were taken with the DS-Qi1 (Nikon, Tokyo, Japan) at 510 nm wavelength. Alternate excitation at 340 and 380 nm wavelengths were filmed using a Polychrome V monochromator, mounted on a Nikon TE2000 inverted microscope (with 20x Sfluor objective; N.A. 0.5). After a baseline period of ca. 2 min, ligands (as shown in figures) were superfused onto the cells, using a gravity-driven perfusion system, which was controlled by ALA VC3 8 valve control system. Fluorescence intensities of ratio images (340/380 nm) on regions of interest (ROI) (including the whole cell) were quantified by NIKON NIS-Elements software after subtraction of background. To distinguish between neuronal and non-neuronal cells, a high potassium (high K+) solution was used to depolarize the cells at the end of the superfusion protocol.
4
Quantification of DREADDs Expression in MCH Neurons
5
Immunofluorescence Staining and Imaging Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded at an equal density on coverslips and fixed with 4% paraformaldehyde. Cells were washed four times with 1× PBS and permeabilized with 0.2% Triton X-100 in PBS for 5 min and then postfixed with 1% paraformaldehyde and 0.01% Tween 20 for 30 min. Cells were blocked for 5 min with 3% BSA/PBS followed by incubation of corresponding primary antibody in 3% BSA/PBS for 1 h at room temperature. Prior to incubation with secondary antibody in 3% BSA/PBS for 1 h at room temperature, cells were washed three times with 1% Triton X-100 in PBS. Cells were then incubated with 0.15 µg/ml DAPI in 1× PBS for 1 min, washed three times with 1× PBS, mounted with fluorescence mounting medium (9 ml of glycerol [BP229-1; Fisher Scientific], 1 ml of 1× PBS, and 10 mg of p-phenylenediamine [PX0730; EMD Chemicals]; pH was adjusted to 8.0–9.0 using carbonate-bicarbonate buffer [0.2 M anhydrous sodium carbonate, 0.2 M sodium bicarbonate]) and sealed. At least 200 cells per coverslip were counted. Images were obtained at room temperature using a Nikon ECLIPSE 90i microscope with a 20×/0.17 objective (Nikon DIC N2 Plan Apo) equipped with a camera (DS-Qi1). Images were acquired using NIS-Elements AR software and processed using ImageJ.
6
Quantifying L-type Ca2+ Channel Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To evaluate L-type Ca2+ channel expression, cells were fixed in 4% PFA at room temperature, permeabilized with 0.2% Triton X-100 and blocked with 20% normal goat serum (Vector, Labs Burlingame, CA, USA) for 1 h at room temperature. Then cells were incubated with rabbit anti-human L-type α1C subunit (CaV1.2) (1:100, Santa Cruz Biotechnology)23 (link) and mouse anti-human osteocalcin (1:50 Santa Cruz Biotechnology), washed and incubated with Alexa-fluor-488 (green) goat anti-mouse and Alexa-fluor 647 (red) goat anti-rabbit respectively (Molecular Probes). Fluorescence was acquired with a Nikon Di-U upright microscope equipped with a Nikon DS-Qi1 digital CCD camera.
7
Immunofluorescent Analysis of L-type Ca2+ Channels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To evaluate L-type Ca2+ channel expression, cells were fixed in 4% PFA at room temperature, permeabilized with 0.2% Triton X-100 and blocked with 20% normal goat serum (Vector, Labs Burlingame, CA, USA) for 1 h at room temperature. Then cells were incubated with rabbit anti-human L-type -type α1C subunit (CaV1.2) (1:100, Santa Cruz Biotechnology), washed and incubated with Alexa-fluor-488 (green) goat anti-mouse (Molecular Probes). Fluorescence was acquired with a Nikon Di-U upright microscope equipped with a Nikon DS-Qi1 digital CCD camera.
8
Patch Clamp Analysis of GABA-A Receptor Mutants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole-cell patch clamp recordings were made from visually identified cortical layer 2/3 neurons in acutely prepared 300-µm thick coronal brain slices (using IR/DIC microscopy) from GABAA-R α4−/− mice and α4+/+ male littermates using a Nikon Eclipse FN1 microscope equipped with a 4× objective and a 40× water immersion objective and a high-sensitivity monochrome digital camera (DS-Qi1, Nikon). Pyramidal cells were selected based on pyramidal shape, apical dendrite, and distance (200–300 µm) from pia. Standard whole-cell patch clamp recordings were made in either the voltage or current clamp configuration using a Multiclamp 700 amplifier (Molecular Devices, Sunnyvale, CA) and pClamp 10.2 software (Molecular Devices). Data were acquired at 10 kHz, filtered at 2.2 kHz, and digitized using a Digidata 1440A A/D interface (Molecular Devices). All voltage clamp experiment recordings were performed at −70 mV; neurons having resting membrane potential greater than −45 mV were omitted from the study. Liquid junction potential (+3.8 mV) was not corrected. Series resistance was less than 25 MΩ following membrane rupture and with whole-cell configuration established; data were excluded if resistance changed by more than 20% over the course of the recording. For analysis of sIPSC decay times, currents were fit with a monoexponential function and the 80–20% decay time recorded.
9
Rat Brain Tissue Preparation and Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rats were anesthetized via an intramuscular injection of a ketamine (90.1 mg/kg body weight), xylazine (2.8 mg/kg body weight), and acepromazine (0.72 mg/kg body weight) cocktail and then transcardially perfused with 0.9 % sterile saline (pH 7.4) followed by 4 % paraformaldehyde (PFA) in 0.1 M borate buffer (pH 9.5). Brains were dissected from the skull and post-fixed in PFA with 15 % sucrose for 24 h, and then flash-frozen in isopentane cooled in dry ice. Brains were then stored at −80 °C. On a freezing microtome, brains were sectioned to 30 μm thickness and sections were collected in 5-series, stored in antifreeze solution at −20 °C, and thereafter immunohistochemistry was performed to visualize viral expression as described in Supplementary Materials . Photomicrographs for confirmation of AAV injection sites and fiber optic cannulae placement within the dorsal dentate gyrus of the hippocampus were captured using a Nikon 80i camera (Nikon DSQI1, 1280X1024 resolution, 1.45 megapixel) under epifluorescence. 70 % of animals were included in data analysis, taking injection site, cannulae placement, and acquisition of quality signal during photometry recordings into consideration.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!