The following procedures were performed at room temperature. Neural cells were fixed with 3.2% paraformaldehyde for 30 minutes and permeabilized for 5 minutes with 0.1% Triton‐x‐100 in PBS. Cells were then treated with Dako Protein Block for 25 minutes to prevent nonspecific antibody binding. Following this, neural cells were incubated with MAP2 (Santa Cruz, cat. no. SC‐74421) and GFAP (Sigma, cat. no. G3893) mouse anti‐human, primary antibodies. After washing the cells 3× with Dako Washing Buffer (WB), appropriate Alexa Fluor‐conjugated secondary antibodies (Invitrogen) were added to cell culture wells; incubation time was 45 minutes. All antibody dilutions were performed according to manufacturers' instructions. Samples were then washed once more with WB and incubated with DAPI (Sigma Aldrich) for 3 minutes. The immunofluorescent cells were visualized with Leica DM IRB inverted microscope system (Leica, Germany) equipped with the Retiga 4000R camera (Qlmaging, Canada), which was controlled with Openlab software version 5.0.2 (Perkin‐Elmer).
Dm irb inverted microscope system
Manufactured by Leica
Sourced in Germany
The Leica DM IRB is an inverted microscope system designed for a variety of laboratory applications. The core function of the Leica DM IRB is to provide high-quality optical imaging and observation capabilities for samples placed on the microscope's stage.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Dako protein block, by Agilent Technologies (1 mentions)
Glial fibrillary acidic protein (gfap), by Merck Group (1 mentions)
Ve cadherin, by BD (1 mentions)
Matrigel, by Corning (1 mentions)
Dmirb inverted microscope, by Leica (1 mentions)
Lsm 510 meta laser, by Zeiss (1 mentions)
Ve cadherin cd144, by Santa Cruz Biotechnology (1 mentions)
Prolong gold antifade mounting medium, by Thermo Fisher Scientific (1 mentions)
α sm actin, by Agilent Technologies (1 mentions)
Alexa fluor 488 or 546 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
5 protocols using dm irb inverted microscope system
1
Immunofluorescent Staining of Neural Cells
2
Immunofluorescence Analysis of iECs
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The following procedures were all performed at room temperature. iECs were fixed with 3.2% paraformaldehyde for 30 min and permeabilized for 5 min with 0.1% Triton-x-100 in PBS. The cells were then treated with Dako Protein Block for 25 min in order to prevent nonspecific antibody binding. Following this, iECs were incubated with the following mouse anti-human, primary antibodies: VE-Cadherin (BD Biosciences) (1 hr) and VWF (R&D Systems) (3 hrs). After washing the cells 3× with Dako Washing Buffer (WB), the appropriate Alexa Fluor-conjugated secondary antibodies (Invitrogen) were added to cell culture wells; the incubation time was 45 minutes. All antibody dilutions were performed according to manufacturers’ instructions. Samples were then washed once more with WB and incubated with DAPI (Sigma Aldrich) for 3 minutes. The immunofluorescent cells were visualized with Leica DM IRB inverted microscope system (Leica, Germany) equipped with a digital camera Retiga 4000R (Qlmaging, Canada), which was controlled with Openlab software version 5.0.2 (Perkin-Elmer).
3
Visualizing Endothelial Cell Tube Networks
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Matrigel (Corning) was thawed overnight at 4°C. The following morning, matrix coating was added to 12-well cell culture plates, which were incubated for 30 min at 37°C and 5% CO2. iECs were seeded at a density of 2.75 × 105 cells per well and incubated for 6 hrs in VascuLife EnGS medium (LifeLine). After the incubation period, the cells were treated with the cell permeable dye Calcein-AM (2 μg/mL) and incubated for 30 min at 37°C and 5% CO2. Afterwards, the 12-well cell culture plates were ready for tube network visualization under the Leica DM IRB inverted microscope system (Leica, Germany) equipped with a digital camera Retiga 4000R (Qlmaging, Canada).
4
Microscopy Imaging Protocols for Cell Analysis
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Confocal images were acquired using a Zeiss LSM 510 META laser scanning microscope system (Zeiss, Oberkochen, Germany). By varying the detectors' pinhole width, the observed fluorescence was localized to an area of known tissue thickness, and the field depth of the transmitted DIC images was adjusted. Scale bars were integrated into the image during acquisition. Epifluorescent images were acquired on a Leica DM IRB inverted microscope system (Leica, Germany) equipped with a digital camera Retiga 4000R (Qlmaging, Surrey, Canada), which was controlled with Openlab software version 5.0.2 (Perkin-Elmer, Waltham, MA). Scale bars were calibrated to each objective magnification and added after acquisition. Light microscopy images were acquired with a Nikon D100 (Nikon, Tokyo, Japan) digital SLR camera on a Leica DM IRB inverted microscope.
5
Immunofluorescence Analysis of mES Cells
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