Atto643 (NHS-Atto643, Atto-Tec) was conjugated to goat anti-rabbit IgG (Sigma) in 100 mM NaHCO3 using 5-fold molar excess of dye for 2 h at RT, purified using Zeba Spin desalting columns with 40 MWCO (Thermo Fisher Scientific) and stored in PBS containing 0.2 % sodium azide. Goat anti-rabbit CF568 (Biotium, 10 µg/ml) and goat anti-mouse Atto643 (custom labeled, 10 µg/ml) were applied to samples in blocking buffer for 2 h at RT. Following washing in PBS (3x, 10 min each), immunostained samples were reacted with crosslinking reagent AcX (Thermo Fisher Scientific) dissolved at a concentration of 1 μg/ml in PBS overnight at RT. After washing away excess AcX (3x, 10 min each, PBS) samples were gelled as previously described61 (link),62 (link). Briefly, coverslips were flipped on 120 µl proExM monomer solution (8.55% sodium acrylate, 2.5% acrylamide, 0.15% bis-acrylamide, 0.2% ammonium persulfate, 0.2% tetramethylethylenediamine) pipetted on parafilm and gelled in a humidified atmosphere at 37 °C for 2 h. Gels were cut to a SIM card shape for the identification of gel orientation and subjected to digestion with 8 U/ml Proteinase K (Thermo Fisher Scientific) in proExM digestion buffer (50 mM Tris pH 8.0, 1 mM EDTA, 0.5% Triton X-100, 0.8 M guanidine HCl) in a 6-well cell culture chamber (3 ml volume) overnight at RT. Digested gels were expanded to the final size by repeated incubations (5-6 times, 15 min each) in double distilled water. Samples were transferred to imaging chambers coated with Poly-D lysine (Merck, high precision glass bottom, 1.5#). Imaging was performed on a LSM700 laser scanning confocal microscope (Zeiss) using a 63X 1.2 NA water objective, equipped with 561 nm and 640 nm DPSS laser lines. Z-Stacks were corrected for chromatic aberration applying elastic transformations based on images of Tetraspeck beads acquired under the same imaging conditions. Image brightness and contrast were linearly adjusted. Confocal images were acquired with ZEN 12.0.1.362 (Zeiss).
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