Cells were cultured in a four-chamber slide (Nunc, Naperville, IL, USA) and fixed with 4% paraformaldehyde for 20 min at RT. After fixation, cells were washed three times with cold PBS and permeabilized with 0.2% Triton X-100 for 10 min, and then blocked with 1% BSA-PBS for 1 h at RT. Cells were then incubated overnight at 4 °C with primary Abs that had been diluted in 1% BSA-PBS. The cells were washed three times with cold PBS, and Alexa Fluor 488- or Alexa Fluor 594-conjugated secondary Abs (Invitrogen) were added for 1 h. The cells were washed three times with cold PBS and mounted with Vectashield mounting solution (Vector Laboratories, Burlingame, CA, USA) and observed under a confocal microscope.
Four chamber slide
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Four-chamber slide is a laboratory equipment used to hold and observe samples. It features four separate compartments, allowing users to conduct multiple experiments or observations simultaneously. The slide is designed to provide a consistent and controlled environment for sample analysis.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield mounting solution, by Vector Laboratories (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Dapi containing medium, by Merck Group (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Q color 5 digital camera, by Olympus (1 mentions)
Bx41 microscope, by Olympus (1 mentions)
Fluoview fv10i confocal microscope, by Olympus (1 mentions)
4 protocols using four chamber slide
1
Immunofluorescence Staining of Cultured Cells
2
Immunofluorescent Detection of NFATc1
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Immunofluorescent detection of NFATc1 was performed according to the method described by Hasega et al. 25 with minor modifications. Briefly, cells were plated at less than 50% confluence (a density of 5 × 104 cells/chamber) in a four‐chamber slide (Nunc, Rochester, NY, USA), grown overnight, and treated with vehicle or drugs for 24 h in complete α‐MEM. After fixation with 4% paraformaldehyde for 10 min at room temperature, cells were permeabilized with 0.2% Triton X‐100 for 15 min and then blocked with 5% bovine serum albumin at room temperature for 1 h. After washing and blocking, anti‐NFATc1 antibody was added at a dilution of 1 : 100 and the fixed cells were incubated at 4 °C overnight. NFATc1 signals were detected with Alexa Fluor 488‐conjugated secondary antibody (Invitrogen) at a dilution of 1 : 500 for 1 h, after which the fixed cells were mounted in DAPI‐containing medium (Sigma).
3
Scratch Wound Healing Assay in Myoblasts
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The myoblasts were plated in four-chamber slides (Thermo Fisher Scientific) to 80% confluency in growth medium. A scratch line was drawn through the cell monolayer using a 200-μl pipette tip. Lifted myoblasts were washed away with growth medium and the medium was replaced with growth medium with paxilline or vehicle.19 (link) The myoblasts were cultured at 37 °C, 5% CO2, for 6 h and the scratch injury area was captured at room temperature using BX41 microscope (Olympus, Melville, NY, USA) equipped with Plan N × 10/0.25 objective lens and Qcapture software (Surrey, BC, Canada) linked to a Q-Color5 digital camera (Olympus). The area of scratch injury was measured using Adobe Photoshop CS3 (San Jose, CA, USA).
4
Immunofluorescence Staining of NFATc2
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Five thousand cells were seeded on the four chamber slides (Thermo Fisher Scientific) one day prior to the immunofluorescence staining. After cell permeabilization and blocking, cells were probed with NFATc2 primary antibody overnight, then with Alexa Fluor 594 dye-conjugated secondary antibody and DAPI (blue-green) for confocal laser scanning. Confocal laser scanning microscopy was performed using a Fluoview FV10i Confocal Microscope (Olympus, Tokyo, Japan) and images were captured with 60X oil objective under different gain settings. The 559 nm laser diode was used to capture NFATc2 staining, and the 405 nm laser diode was used to capture the DAPI nuclear stain. Image acquisition and further adjustment of brightness was performed using an Olympus FluoView FV10ASW Version 4.2a software. Fluorescent images of cells were taken as single channel images and then converted to overlay images and all images were saved in TIFF format.
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