E12.5 ventral spinal cord horns were extracted, as described above. Tissue pieces were seeded onto the bottom of a 35 mm Ø glass plate (NuncTM), on top of a thin layer of Matrigel™ previously mixed with recombinant VEGF (50 ng/mL; Peprotech), LIF (1 ng/mL~104 U/mL; Merck Millipore, Germany), GDNF (30 ng/mL, Peprotech), or Angiopoietin I (100 ng/mL; R&D Systems, USA), or a combination of these. Angpt1 was complexed overnight with a 1:100 dilution of an AntiHis antibody (R&D Systems) before adding it to the MatrigelTM. After that, a 20 µL drop of the corresponding Matrigel™ mixture was placed on top of the explant and left at 37 °C for jellification. After 15’, MN culture media without factors (BDNF, CTNF, and GDNF) was added to each plate. Photomicrographs were taken 24 h after seeding with an Olympus IX71 inverted microscope and a Hamamatsu Orca Flash 4.0 camera.
Ix71 inverted microscope
Manufactured by Hamamatsu Photonics
The IX71 inverted microscope is a high-quality optical instrument designed for various applications. It features a sturdy, ergonomic design and offers a range of capabilities to support research and analysis activities. The IX71 is capable of providing clear, high-resolution images of specimens under observation.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant vegf, by Thermo Fisher Scientific (1 mentions)
Anti his antibody, by R&D Systems (1 mentions)
Orca flash 4.0 camera, by Hamamatsu Photonics (1 mentions)
Orca r2 monochrome camera, by Hamamatsu Photonics (1 mentions)
Orca r2 ccd, by Olympus (1 mentions)
1.35 na oil objective, by Olympus (1 mentions)
Orca r2 c10600 10b digital ccd camera, by Hamamatsu Photonics (1 mentions)
C4742 80 12ag, by Hamamatsu Photonics (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Coolsnap hq2 camera, by Nikon (1 mentions)
Orca r2 camera, by Hamamatsu Photonics (1 mentions)
Nis elements imaging software, by Nikon (1 mentions)
Ccd camera, by Hamamatsu Photonics (1 mentions)
Zen software, by Zeiss (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Click it edu alexa fluor 488 imaging kit, by Thermo Fisher Scientific (1 mentions)
D1306, by Thermo Fisher Scientific (1 mentions)
49 6 diamidine 2 phenyl indole dapi, by Thermo Fisher Scientific (1 mentions)
7 protocols using ix71 inverted microscope
1
Ventral Spinal Cord Explant Culture
2
Quantifying Neuronal Differentiation in SK-N-SH Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Five thousand SK-N-SH cells were seeded per well in 12-well plates and chemically treated the next day. After treatment, phase-contrast images were recorded (ten fields of view per well) with an Olympus IX71 inverted microscope and a Hamamatsu Orca R2 monochrome camera and randomised for blind analysis. The length of the neurites in each image was measured using the NeuroJ plugin on ImageJ76 . Only neurites longer than the cell body were measured and an average neurite length per field was calculated.
3
In utero and ex utero time-lapse imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In utero time lapse: adult hermaphrodite worms were anesthetized as previously described (Kirby et al., 1990 (link); McCarter et al., 1999 (link)) and mounted between a 22 × 30 mm coverslip and a thin 3% agarose pad on a slide (Figures 2, A and B , 3C , 4A , and 6A ). Ex utero time lapse: adult hermaphrodite worms were placed in 10 μl of 0.8X egg salts (1× egg salts: 118 mM NaCl, 40 mM KCl, 3.4 mM MgCl2, 3.4 mM CaCl2, and 5 mM HEPES, pH 7.4; Carvalho et al., 2011 (link)) on a 22 × 30 mm coverglass where the embryos were dissected out and the remaining worm carcasses were carefully removed. The coverslip was then inverted onto a slide that had coverslips attached as spacers to prevent squashing the embryos (Figures 1, A and C , 2D , and 5, A and C ). Images in Figures 2D , 3C , and 4A were captured with a PerkinElmer-Cetus Ultraview Spinning Disk Confocal equipped with an Orca R2 CCD and an Olympus 100×/NA 1.35 oil objective. Images in Figures 1, A and C , 2, A and B , and 5, A and C ) were captured with an Olympus IX71 inverted microscope with a 60×/NA 1.42 Plan-Apo objective and equipped with a Hamamatsu Orca R2 C10600-10B digital CCD camera. Images in Figure 6A were captured with a PerkinElmer-Cetus Ultraview Spinning Disk Confocal equipped with an ORCA Flash 4.0 CMOS camera and an Olympus 100×/NA1.35 oil objective.
4
Immunofluorescent Detection of Primary Cilia
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Confluent cell monolayers exposed overnight to the various experimental conditions were rinsed twice with phosphate-buffered saline (PBS) and fixed for 10 min in a freshly prepared solution of paraformaldehyde (4%) and sucrose (2%). Cells were then washed three times with PBS, blocked for 30 min with BSA (1%) in PBS, and incubated for 60 min with an anti-acetylated-α-tubulin antibody (Santa Cruz Biotechnology) to identify primary cilia (Raychowdhury et al., 2005 (link)). Anti-mouse IgG FITC-coupled (Invitrogen) was used as secondary antibody. Cells were counter-stained with DAPI to locate cell nuclei and mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Cells were viewed under an Olympus IX71 inverted microscope connected to a digital CCD camera C4742-80-12AG (Hamamatsu Photonics KK, Bridgewater, NJ). Images were collected and analyzed with the IPLab Spectrum acquisition and analysis software (Scanalytics, Viena, VA), running on a Dell-NEC personal computer.
5
Imaging Neurons with EB3 Comets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence stained images were acquired with an Olympus IX-71 inverted microscope equipped with a CoolLED fluorescent light source, a Hamamatsu ORCA-R2 camera, and MetaMorph software. 10 × 0.4 N.A., 40 × 0.95 N.A., or 60 × 1.35 N.A. Plan Apochromat objective lenses were used to collect fluorescence images.
Live cell imaging was performed on a Nikon Eclipse-Ti inverted microscope equipping with a TIRF illuminator, a built-in Perfect Focus system, and a Tokai Hit TIZHB live cell chamber. Images were acquired using a 60 × 1.49 N.A. Plan Apochromat objective lens, a 561 nm DPSS laser, a Photometrics CoolSNAP HQ2 camera, and Nikon NIS-Elements imaging software. Images were acquired every 500 milliseconds over a 2-minute period. Only the neurons with clear EB3 comets were imaged.
Live cell imaging was performed on a Nikon Eclipse-Ti inverted microscope equipping with a TIRF illuminator, a built-in Perfect Focus system, and a Tokai Hit TIZHB live cell chamber. Images were acquired using a 60 × 1.49 N.A. Plan Apochromat objective lens, a 561 nm DPSS laser, a Photometrics CoolSNAP HQ2 camera, and Nikon NIS-Elements imaging software. Images were acquired every 500 milliseconds over a 2-minute period. Only the neurons with clear EB3 comets were imaged.
6
Visualizing Astrocyte Vesicular Fusion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
An Olympus total internal reflection fluorescence (TIRF) microscope was used to detect vesicular fusion events in astrocytes expressing VNUT–eGFP or CD63‐mKate, as described in detail previously (Angelova et al., 2015 ; Kasymov et al., 2013 ). Fluorescence was excited at 488 nm and collected at 500–530 nm for eGFP and excited at 488 nm and collected at 600–660 nm for mKate. The imaging setup was equipped with a high‐NA oil‐immersion objective (60×, 1.65 NA), an Olympus IX71 inverted microscope and a Hamamatsu CCD camera. Images were acquired using Olympus Cellt ^
7
Quantifying Cell Proliferation in Wound Healing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To assess proliferation in confluent and wound-edge cells, the Click-iT EdU AlexaFluor488 Imaging Kit (C10337; Life Technologies, Burlington, Ontario, Canada) was used according to the manufacturer's protocol. Briefly, 50% of the media was replaced with fresh media containing 20 mM EdU (final concentration 10 mM EdU) and cells were returned to the incubator for 2 hours. Coverslips were fixed with 4% paraformaldehyde (43368; Alfa Aesar, Ward Hill, MA) for 15 minutes, permeabilized with 0.5% Triton-X for 20 minutes, incubated with Click-iT reaction cocktail for 30 minutes, and then incubated with 49,6-diamidine-2-phenylindole (DAPI) for 30 minutes (1:50000, D1306; Invitrogen Calsbad, Ca). Coverslips were mounted with FluorSave (34789; Calbiochem, Darmstadt, Germany). Widefield (10Â) images were captured with an Olympus IX71 inverted microscope equipped with a Hamamatsu OrcaR2 12-bit camera. Using ImageJ software, cells were counted separately in DAPI and EdU images, and data were expressed as percentage of EdU-positive cells.
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!