Visualizing Connexin Protein Localization

Control cells or cells expressing GFP- or FLAG-tagged Cx30.3, Cx30.3 mutants or co-expressing Cx30.3 mutants together with RFP-tagged Cx30.3, Cx26, Cx30, or Cx43 were plated on sterile glass coverslips (ThermoFisher, Cat# 1254580). Cells were grown to ∼80% confluence, rinsed twice with 1x phosphate-buffered saline (PBS) (ThermoFisher, Cat# 10010-023) and fixed in ice-cold 80% methanol/20% acetone (v/v) solution for 15 min at 4°C. After rinsing in PBS, cells were blocked with 2% (w/v) bovine serum albumin (BSA; MilliporeSigma, Cat# A2153) for 30 min at room temperature. Cells were labelled for 45 min at room temperature with a 1:200 dilution of the following primary antibodies: mouse monoclonal anti-FLAG (MilliporeSigma, Cat# F3165), anti-PDI (Enzo Life Science, Cat# ADI-SPA-891F), anti-E-cadherin (BD Biosciences, Cat# 610182) or rabbit monoclonal anti-Cx43 (MilliporeSigma, Cat# C2619), anti-FLAG (Cell Signaling, Cat# 14793) and anti-BiP/GRP78 (MilliporeSigma, Cat# G8918). Cells were then washed with PBS and incubated for 45 min in the secondary antibody Alexa Fluor 555-conjugated goat monoclonal anti-mouse (ThermoFisher, Cat# A32727) or anti-rabbit (ThermoFisher, Cat# A32732) IgG (1:800 dilution in 2% BSA), or in some cases, AlexaFluor 488-conjugated donkey anti-mouse (ThermoFisher, Cat# A21202) or donkey anti-rabbit (ThermoFisher, Cat# A21206) IgG (both used at 1:800 dilution). All antibodies were diluted as indicated in blocking solution. All coverslips were then stained with Hoechst 33342 (1:1000 dilution in distilled water; ThermoFisher, Cat# H3570) for 1 min to demarcate the nuclei and mounted onto glass microscope slides using Airvol mounting solution. In some cases, cells were only labelled with Hoechst 33342.
Cells were imaged on a Zeiss LSM800 confocal microscope equipped with Zen2.3 software (Zeiss International, Oberkochen, Germany, www.zeiss.com) and a ×63 oil immersion objective lens. When imaging samples for direct comparisons, all images were acquired and presented using identical imaging conditions (laser strength, pinhole, and contrast) to ensure fluorescence intensity could be directly compared. For gap junction quantification, a third-party investigator blinded to the treatment quantified the percent of gap junction forming cell pairs. Gap junction plaques were defined as a linear or punctate green fluorescence signal of approximately 0.2 μm in length, situated at the interface between two transfected cells. Experiments were repeated at least three times, with 30 or more cells or cell-cell interfaces analyzed for each group in each repeat.