Alexafluor555 conjugated donkey anti goat antibody

The cells were placed on glass slides, fixed in acetone for 5 min at room temperature (15–25° C), and dried completely before staining. Cells were incubated overnight at 4° C with an anti-SFRP4 antibody (Thermo Fisher Scientific) and anti-p16^ink4a antibody (Abcam, Cambridge, UK) diluted at 1:100 in phosphate-buffered saline (PBS). After washing thrice with PBS, the slides were incubated with Alexa Fluor 488 conjugated goat anti-rabbit antibody and AlexaFluor555 conjugated donkey anti-goat antibody (Thermo Fisher Scientific) diluted at 1:2000 in PBS for 1 h at room temperature. After incubation, the slides were washed thrice with PBS and counterstained for nuclear visualization using ProLong Gold Anti-fade Mountant (Thermo Fisher Scientific) containing 4ʹ,6-diamidino-2-phenylindole.

Cells were placed on glass slides and fixed in 4% paraformaldehyde at room temperature (20–25 °C) for 10 min. The cells were incubated with anti-EPDR1 antibody (PA5-140254, Thermo Fisher Scientific) and anti-p16ink4a antibody (ab211542; Abcam, Cambridge, UK) diluted 1:100 in phosphate-buffered saline (PBS) at 4 °C overnight. After three washes with PBS, the cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit antibody and Alexa Fluor 555-conjugated donkey anti-goat antibody (each from Thermo Fisher Scientific), diluted 1:2000 in PBS at room temperature for 1 h. After incubation, the slides were washed three times with PBS and counterstained for nuclear visualization using ProLong Gold Antifade Mountant (Thermo Fisher Scientific) containing 4′,6-diamidino-2-phenylindole.

Cells were placed on glass slides, fixed in acetone at room temperature (15–25 °C) for 5 min, and dried completely before staining. The cells were incubated overnight at 4 °C with an anti-cathepsin F antibody (PA5-87,925, Thermo Fisher Scientific) and anti-IL-6 antibody (ab6672, Abcam, Cambridge, UK) diluted 1:100 in phosphate-buffered saline (PBS). To confirm the specificity of the immunostaining, fetal bovine serum diluted to the same concentration was used instead of the primary antibody as a negative control. After washing three times with PBS, the slides were incubated with Alexa Fluor 488-conjugated goat anti-rabbit antibody and AlexaFluor555-conjugated donkey anti-goat antibody (Thermo Fisher Scientific) diluted 1:2000 in PBS for 1 h at room temperature. After incubation, nuclei were washed three times with PBS and counterstained for the visualization of nuclei using ProLong Gold Anti-fade Mountant containing 4′,6-diamidino-2-phenylindole (Thermo Fisher Scientific).