1. NRK cells plated on 35 mm glass-bottom dishes (MatTek Corporation, Ashland, MA) pre-coated with poly-d-lysine are grown in Dulbecco’s modified Eagle’s medium (Mediatech, Inc., Manassas, VA) supplemented with 10 % FBS in a humidified 5 % CO 2 incubator at 37 °C. Cells should be ~70–90 % confluent to be used for the experiment.
2. Buffered solutions. Hanks’ Balanced Salt Solution (HBSS) with calcium and magnesium, no phenol red, 1×, pH 7.0 (LifeTechnologies). Phosphate-Buffered Saline (PBS), pH 7.0.
3. Antibodies. Primary antibody: rabbit polyclonal anti-Cx43 (dilution 1:400; Sigma, Cat. # C6219). Secondary antibodies: Alexa 488-FluoroNanogold goat anti-rabbit (dilution 1:100; Nanoprobes, Inc., Yaphank, NY) or FITC goat anti-rabbit (dilution 1:100; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA).
4. Fixative. Prepare a 4 % (w/v) paraformaldehyde (PFA) solution in PBS. To make a volume of 50 ml: heat up 40 ml of ddH2O at 60 °C with a stirring hot plate in a fume hood; turn off the hot plate and dissolve 2 g of PFA prills (Electron Microscopy Sciences) and while stirring add 25 μl of 5 N NaOH. Filter the solution in a graduated cylinder using a filter paper. Add 5 ml of 10× PBS. Adjust pH to 7.4. Adding ddH2O bring the volume to 50 ml.
4 % PFA/PBS + 0.1 % glutaraldehyde: add 200 μl of 25 % glutaraldehyde solution in 50 ml of freshly prepared PFA.
2 % PFA/PBS: follow same procedure as 4 % PFA/PBS adjusting the quantity of PFA prills according to the desired concentration.
2 % PFA/PBS + 0.1 % glutaraldehyde: add 200 μl of 25 % glutaraldehyde solution in 50 ml of freshly prepared PFA.
2 % glutaraldehyde/PBS: add 1 ml of 25 % glutaraldehyde solution in 11 ml PBS (1×), to obtain a total volume of 12 ml.
5. Quenching solution. 50 mM glycine in PBS. Dissolve 188 mg of glycine in 50 ml of PBS (1×).
6. Blocking/permeabilizing buffer. 1 % Bovine Serum Albumin (BSA), 5 % Normal Goat Serum (NGS), 0.1 % Triton X-100, 20 mM glycine, 1 % fi sh gelatin in PBS. Dissolve 0.5 g of BSA, 2.5 ml of NGS, 50 μl of Triton X-100, 0.075 g of glycine, and 500 μl of fish gelatin in 50 ml of PBS (1×).
7. Working buffer. Dilute blocking buffer 1:10 in PBS (1×).
8. Gold enhancement EM kit (Nanoprobes, Inc., Yaphank, NY). The mixture should be prepared just before use.
9. Ice-cold ethanol solutions: 20–50–70–90–100 %.
10. LR White resin (London Resin Company, Berkshire, England).
11. Aclar Embedding Film (Electron Microscopy Sciences).
12. 200 mesh copper grids (Electron Microscopy Sciences).
13. Diatome diamond knife for ultrathin sections (Electron Microscopy Sciences).