Prolong antifade mountant

For cytoskeleton staining, cells were incubated with Alexa Fluor 488 phalloidin (Thermo Fisher), a high-affinity filamentous actin (F-actin) probe conjugated to green-fluorescent Alexa Fluor 488 dye (Thermo Fisher) for 1 h. The stained cells were mounted with DAPI-containing ProLong Antifade Mountant (Thermo Fisher) and observed using a Cytation Confocal Imaging Reader (Biotek/Agilent). To quantitatively analyze fluorescent intensities, cells of interest and regions of interest (ROI) were selected and measured for mean fluorescence intensity using ImageJ software. Corrected Total Cell Fluorescence (CTCF) was calculated based on: CTCF = Integrated Density − (Area of Selected Cell × Mean Fluorescence of Background readings).

Transfected cells were fixed with 4% paraformaldehyde for 20 min at RT, washed with PBS, and mounted with ProLong Antifade Mountant (Thermo Fisher Scientific, Rockford, IL, USA) for imaging. Samples were visualized using a Zeiss LSM 700 confocal microscope using a Plan-Apochromat 63x/1.4 Oil DIC M27 objective. Zeiss ZEN 2010 program was used to control imaging specifications. The gap junction area was determined using ImageJ by tracing the gap junction area with a free hand tool followed by quantification using the measure tool. ImageJ software was also used to analyze co-localization data.
In the case of immunofluorescence, transfected cells were fixed with ice-cold 100% methanol for 10 min at RT. Cells were blocked using PBS supplemented with 2% BSA for 1 h at RT. Primary antibody, rabbit anti-HIS (Bethyl Laboratories Inc., Montgomery, TX, USA) at 1:1000 concentration, was diluted in PBS with 0.1% BSA and applied to cells for 1 h at RT. Cells were then incubated in the secondary antibody solution containing 2 μg/mL of Alexa Fluor 568 goat anti-mouse (Thermo Fisher Scientific, Rockford, IL, USA) and 2 μg/mL of Alexa Fluor 488 donkey anti-rabbit (Thermo Fisher Scientific, Rockford, IL, USA) in PBS with 0.1% BSA for 1 h at RT. Cells were washed in PBS and mounted with FluoroshieldTm (Sigma-Aldrich Chemie GmbH, Munich, Germany).

Cultured DRG neurons were fixed with 3% paraformaldehyde and 0.1% glutaraldehyde in PBS for 20 min and permeabilized with 0.1% Triton X-100 in PBS for 10 min. DRG neurons were then incubated with a high-affinity F-actin probe, Alexa Fluor 488^TM phalloidin (1:40, ThermoFisher Scientific # A12379), in PBS for 20 min at RT, followed by a 5 min incubation with the nuclear counterstain DAPI (ThermoFisher Scientific # 62248). Samples were preserved using ProLong Antifade Mountant (ThermoFisher Scientific # P36961). Images were acquired on a Zeiss LSM710 confocal microscope with a 63x/1.4 Plan-Apochromat oil immersion objective. Images were processed using ZEN2010 software (Zeiss) and analyzed using Fiji ImageJ^{103 (link)} (v.2.3.0/1.53q) to enhance contrast and convert to an appropriate format.

Mouse kidney slices from formalin‐fixed and paraffin‐embedded tissue blocks underwent deparaffinization and rehydration using standard protocols. The slices were then treated with cold acetone for 10 min at ‐20 °C and blocked with 1% BSA for 1 h at room temperature (RT). For VCAM‐1 staining, the FITC‐conjugated anti‐VCAM‐1 antibodies (sc‐18864 FITC, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:50 dilution in 1% BSA) were applied to the slides and incubated at RT for 1 h at dark. Nucleus were stained with DAPI. Cover glass was mounted using the Prolong Antifade Mountant (Thermo Fisher Scientific). The fluorescence images of tissues were then acquired using a Zeiss Apotome microscope (Zeiss, Oberkoche, Germany).