Mouse anti–ZO-1 (33-9100) and rabbit ZO-2 (38-9100) antibodies were from GE Healthcare, mouse Myc-Tag (9B11) antibody was from Cell Signaling Technology, rabbit anti-GFP (Ab290) and rabbit anti–N-cadherin (Ab76057) antibodies were from Abcam, and rabbit anti–β-catenin (C2206) antibody was from Sigma-Aldrich. Antibodies were validated by recognizing bands of the predicted size on immunoblots and by cellular immunolocalization where previously reported. Species-specific secondary antibodies for immunofluorescence (Cy2, Cy3, and Cy5 conjugated) and immunoblots (IR-labeled 680 and 790/800 antibodies) were from Jackson ImmunoResearch. Rhodamine phalloidin and Alexa Fluor 647 phalloidin were from GE Healthcare. Halo ocln was labeled using Janelia Fluor 646 (Grimm et al., 2015 (link)) at 1:200 in cell culture media plus serum, followed by four 10-min washes to allow free dye to diffuse out. Cells were otherwise fixed and stained normally.
Protocol detail
Immunostaining of Cell-Cell Junctions
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Alexa fluor 647
Antibodies
Antibody
Cadherin
Cell culture
Cells
Culture media
Immunoblots
Immunofluorescence
Mouse
Phalloidin
Rabbit
Rhodamine phalloidin
Serum
β catenin
Corresponding Organization : National Institutes of Health
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Variable analysis
independent variables
- Mouse anti–ZO-1 (33-9100) antibody
- Rabbit ZO-2 (38-9100) antibody
- Mouse Myc-Tag (9B11) antibody
- Rabbit anti-GFP (Ab290) antibody
- Rabbit anti–N-cadherin (Ab76057) antibody
- Rabbit anti–β-catenin (C2206) antibody
- Rhodamine phalloidin
- Alexa Fluor 647 phalloidin
- Halo ocln labeled using Janelia Fluor 646
- Protein expression levels
- Cellular localization of proteins
- Species-specific secondary antibodies for immunofluorescence (Cy2, Cy3, and Cy5 conjugated) and immunoblots (IR-labeled 680 and 790/800 antibodies) from Jackson ImmunoResearch
- Antibodies were validated by recognizing bands of the predicted size on immunoblots and by cellular immunolocalization where previously reported.
- No negative controls were explicitly mentioned.
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