Cells were fixed for 10 min with 4% (w/v) paraformaldehyde at room temperature. After washing, cells were permeabilized with 0.5% (v/v, PBS) Triton X-100 for 10 min and blocked with 1% (w/v, PBS) bovine serum albumin for 1 h at room temperature. Cells were incubated for 1 h with primary antibodies (1:300) against TMEM16F (Davids Biotechnology, Regensburg, Germany). Binding of the primary antibody was visualized by incubation with appropriate secondary antibodies conjugated with AlexaFluor 488 (1:500, Molecular Probes, Invitrogen). Nuclei were stained with Hoe33342 (0.1 g/mL PBS, Applichem, Darmstadt, Germany) or DAPI. Glass cover slips were mounted on glass slides with fluorescent mounting medium (DAKO Cytomation, Hamburg, Germany) and examined with an ApoTome Axiovert 200 M fluorescence microscope (Zeiss, Germany).
Apotome axiovert 200 m fluorescence microscope
Manufactured by Zeiss
Sourced in Germany
The ApoTome Axiovert 200 M is a fluorescence microscope designed by Zeiss. It is equipped with the ApoTome optical sectioning technology, which enables high-contrast, artifact-free imaging of fluorescent samples. The microscope provides optical sectioning capabilities for improved z-resolution and reduced out-of-focus blur.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Axiocam hrm camera, by Zeiss (2 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (2 mentions)
Hoe33342, by AppliChem (1 mentions)
Fluorescent mounting medium, by Agilent Technologies (1 mentions)
Fluorescence mounting medium, by Agilent Technologies (1 mentions)
β catenin, by Merck Group (1 mentions)
Vectashield with dapi, by Vector Laboratories (1 mentions)
4 protocols using apotome axiovert 200 m fluorescence microscope
1
Immunofluorescence Staining of TMEM16F
2
E-cadherin Immunofluorescence Imaging
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Cells were grown to at least 80% confluence, fixed and stained for E-cadherin. E-cadherin was tagged using a specific antibody (BD Biosciences, Erembodegem, Belgium; mouse, 1:100) and a subsequent fluorescent secondary antibody (Alexa Fluor 488 goat anti-mouse, Invitrogen, Grand Island, NY, USA). Nuclei were counterstained with DAPI. Images were acquired on a Carl Zeiss Apotome Axiovert 200 M Fluorescence Microscope (Carl Zeiss, Jena, Germany) with an Axiocam HRm camera, under a × 40 objective. All experiments were confirmed in three biological replicas.
3
Immunofluorescence Staining of Transfected FRT Cells
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Transfected FRT cells were fixed for 10 min with 4%(w/v) paraformaldehyde as described previously [18 (link)]. In brief, cells were permeabilized and blocked with 2% (w/v, PBS) bovine serum albumin and 0.04% (v/v, PBS) Triton X-100. After 1 h incubation with primary antibody mouse anti-His tag (1:500, Qiagen, Hilden, Germany) at 37 °C, cells were incubated with a secondary donkey anti-mouse antibody conjugated with AlexaFluor 488 (1:1.000, Molecular Probes, Invitrogen). Nuclei were stained with Hoe33342 (0.1 µg/mL PBS, Aplichem, Darmstadt, Germany). β-Catenin (primary rabbit antibody from Sigma-Aldrich (C2206, Deisenhofen, Germany) was visualized using an Alexa 568-labeled secondary antibody. Cells were mounted on glass slides with fluorescence mounting medium (DAKOCytomation, Hamburg, Germany) and examined with an ApoTome Axiovert 200 M fluorescence microscope (Zeiss, Göttingen, Germany).
4
Quantifying E-cadherin Localization at Cell-Cell Junctions
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Cells seeded on top of glass coverslips were washed in PBS, fixed on ice-cold methanol for 20 min and blocked with 3% BSA in PBS for 30 min, at room temperature. E-cadherin was stained with a specific mouse monoclonal antibody (BD Biosciences, Franklin Lakes, NJ, USA), diluted at 1:300 in blocking solution. The Alexa Fluor 488 goat anti-mouse (1:500, Invitrogen, Waltham, MA, USA) was applied as secondary antibody for 1 h in the dark. Coverslips were mounted on slides using Vectashield with DAPI (Vector Laboratories, Burlingame, CA, USA). Images were acquired on a Carl Zeiss Apotome Axiovert 200 M Fluorescence Microscope with an Axiocam HRm camera, and processed with Zeiss Axion Vision 4.8 software. For quantitative purposes, the intensity of fluorescent signals that occur between two contiguous cells (nuclei) was extracted as described by Sanches et al. [21 (link),29 (link)]. Position 1 corresponds to the geometric center of nucleus 1, position 100 is the center of nucleus 2, whereas position 50 represents the plasma membrane. Signal intensity of each position from 1 to 100 was obtained and statistically examined.
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