Digitonin solution

Cells were washed with phosphate-buffered saline (PBS; Oxoid, BR0014G), then fixed with 4% paraformaldehyde in PBS for 20 min and permeabilized with 0.2% Triton X-100 (Sigma Aldrich, T8787) solution or 100 μg/ml digitonin solution (Life Technologies, BN2006) for 20 min, as indicated. Cells were incubated with the appropriate primary antibodies for 1 h, washed 3 times with PBS, and then incubated for 30 min with appropriate Alexa Fluor 488-conjugated (Life Technologies, A21202), Alexa Fluor 555-conjugated (Life Technologies, A31272) or Alexa Fluor 647-conjugated (Life Technologies, A21245) secondary antibodies and then washed again 3 times in PBS. Nuclei were stained with a solution of 1.5 μM of 4′,6-diamidino-2-phenylindole (DAPI; Sigma Aldrich, D9542) in PBS for 5 min. Coverslips were mounted in Fluorescence Mounting Medium (Dako, S3023). Samples were visualized on a TSC SP5 confocal microscope (Leica Microsystems, Germany, Mannheim) installed on an inverted LEICA DMI 6000CS microscope (Leica Microsystems, Germany, Mannheim) and equipped with an oil immersion PlanApo 63X 1.4 NA objective. Images were acquired using the LAS AF acquisition software (Leica Microsystems).

Cells were cultured and treated as previously described.⁶⁵ Briefly, they were washed with PBS, then fixed with 4% paraformaldehyde (Sigma-Aldrich, 47608) in 1X PBS for 20 min and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich, T8787) for 10 min or 100 μg/ml digitonin solution (Life Technologies, BN2006) for 20 min. Permeabilized cells were incubated with the appropriate primary antibodies for 1 h, washed 3 times with 1X PBS, incubated for 30 min with appropriate secondary antibodies and then washed again 3 times in 1X PBS. Nuclei were stained with a solution of 1.5 μM of 4’,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, D9542) in PBS for 5 min. Coverslips were mounted in Fluorescence Mounting Medium (Dako, S3023). Samples were visualized on a TSC SP5 confocal microscope (Leica Microsystems, Germany, Mannheim) installed on an inverted LEICA DMI 6000CS microscope (Leica Microsystems, Germany, Mannheim) and equipped with an oil immersion PlanApo 63 × 1.4 NA objective. Images were acquired using the LAS AF acquisition software (Leica Microsystems).

Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS; Oxoid, BR0014G) for 20 min, washed with PBS) and then permeabilized with digitonin solution (Life Technologies, BN2006) for 20 min. Then, the cells were washed 3 times in PBS. Cells were blocked for 30 min with a 1% BSA (Sigma-Aldrich, A2153) in PBS; after this time, cells were incubated with appropriate primary antibodies for 1 h, washed 3 times, and then incubated with appropriate Alexa Fluor secondary antibodies; subsequently cells were washed 3 times. Nuclei were stained with a solution of 6 μM of 4ʹ,6-diamidino-2-phenylindole (DAPI; Sigma Aldrich, D9542) in PBS for 10 min. Coverslips were mounted in fluorescence mounting medium (Dako, S3023). Samples were visualized on a TSC SP5 confocal microscope (Leica Microsystems, Germany, Mannheim, 5100000750) installed on an inverted LEICA DMI 6000CS (Leica Microsystems, Germany, Mannheim, 10741320) microscope and equipped with an oil immersion PlanApo 63 × 1.4 NA objective. Images were acquired using the LAS AF acquisition software (Leica, 10210).