2.4. Indirect Immunofluorescence

Cells were sparsely seeded on a coverslip and grown to 70% confluence, fixed with 100% ice-cold methanol for 30 min, permeabilised in 0.1% saponin-TBS solution, and blocked in 2% normal goat serum in PBS for 60 min. Cells were then sequentially incubated with the primary polyclonal rabbit anti-v-Jun antibody (Antibodies–online ABIN1109458, cross reactivity for avian and mammalian c-Jun) or primary polyclonal rabbit anti-human c-Fos antibody (Sigma F7799, cross reactivity for mouse, rat, and pig c-Fos, Sigma, Prague, CZ) (1% in PBS for 90 min at room temperature) and Atto 488–labelled goat anti-rabbit IgG secondary antibody (Sigma 18772—0.5% in PBS, 60 min at room temperature in dark, Sigma, Prague, CZ), with extensive washing after each incubation. The negative control staining wascarried out by omitting the respective primary antibody (Figure S2). The coverslips were mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) and analysed using the Olympus AX70 fluorescent microscope equipped with the Olympus DP71 camera system.