Cells were grown on coverslips for immunofluorescence and fixed in 4% PFA in PBS for 20 min at room temperature. Cells were blocked and permeabilized with 1% bovine serum albumin (Sigma) and 0.05% saponin (Sigma) in PBS for 30 min. Fixed cells were then incubated with primary antibodies for 1 h and then for a further 1 h with appropriate secondary antibodies conjugated with a fluorophore (Molecular Probes). The antibodies used and respective dilutions are displayed in Table S1 . Coverslips were finally mounted in MOWIOL mounting medium (Calbiochem). All antibody incubations and washes were done with 1× PBS, 0.5% BSA and 0.05% saponin. To visualize the nuclei, cells were incubated with 4′,6‐diamidino‐2‐phenylindole (DAPI) (Invitrogen) for 5 min. Images were acquired on a Zeiss Observer Z2 widefield microscope, equipped with a Zeiss 506 Mono camera using the 63× 1.4 NA Oil objective or a Zeiss LSM 710 confocal microscope with a Plan‐Apochromat 63× 1.4 NA oil‐immersion objective. Images were processed using ImageJ and Adobe Illustrator 6.0 (Adobe, San Jose, CA, USA) software.
Observer z2 widefield microscope
Manufactured by Zeiss
The Observer Z2 is a widefield microscope designed for versatile laboratory applications. It offers high-quality optics and a robust construction to provide reliable and consistent performance. The core function of the Observer Z2 is to enable wide-field observation and imaging of samples across a range of scientific disciplines.
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Lab products found in correlation
Illustrator 6, by Adobe (2 mentions)
Saponin, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Plan apochromat, by Zeiss (1 mentions)
Plan apochromat 63x 1.4 na, by Zeiss (1 mentions)
Imagej, by Adobe (1 mentions)
2 protocols using observer z2 widefield microscope
1
Immunofluorescence Staining of Cultured Cells
2
Immunofluorescence Staining of Cells
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Cells were grown on coverslips for immunofluorescence and fixed in 4% PFA in PBS for 20 minutes at room temperature. Cells were blocked and permeabilized with 1% bovine serum albumin (Sigma) and 0.05% saponin (Sigma) in PBS for 30 minutes. Fixed cells were then incubated with primary antibodies for 1 hour and then for a further 1 hour with appropriate secondary antibodies conjugated with a fluorophore (Molecular Probes). The antibodies used and respective dilutions are displayed in Supplementary Table 1. Coverslips were finally mounted in MOWIOL mounting medium (Calbiochem). All antibody incubations and washes were done with 1 x PBS, 0.5% BSA and 0.05% saponin. To visualize the nuclei, cells were incubated with DAPI (Invitrogen) for 5 minutes. Images were acquired on a Zeiss Observer Z2 widefield microscope, equipped with a Zeiss 506 Mono camera using the 63x 1.4 NA Oil objective or a Zeiss LSM 710 confocal microscope with a Plan-Apochromat 63x 1.4 NA oilimmersion objective. Images were processed using ImageJ and Adobe Illustrator 6.0 (Adobe, San Jose, CA, USA) software.
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