Labelling of antibodies

Secondary antibodies (goat αRabbit, 111-005-003, AffiniPure or donkey αMouse, 715-005-151, AffiniPure) were diluted 1:10 in labelling buffer (0.2 M NaHCO₃, pH 8.3). Cy3b NHS Ester (PA63101, Life Sciences, Germany) was added to the diluted antibodies in 10-fold molar excess. The samples were incubated for 1 hour at RT. To stop the reaction, 100 mM Tris pH 8.0 was added. Zebra spin desalting columns (8989, Thermo Fisher Scientific) were equilibrated with PBS. The samples were added to the column and centrifuged at 1,000 g for 2 minutes. The filtrate was added to a second column and centrifuged at 1,000 g for 2 minutes.