4 6 diamidino 2 phenylindole dapi containing mounting solution

Fixation was carried out with ice-cold 4% paraformaldehyde (30 min), followed by permeabilization when necessary with 0.1% Triton X-100 on ice (10 min) and blocking in 3% bovine serum albumin (BSA;30 min). Incubation with primary antibodies was carried out in a 3% BSA solution (overnight) at 4 °C. Primary antibodies used were: rabbit anti-SIRT1(H300) (1:400; Santa Cruz, Cat. No. sc-15404), rabbit anti-NF-kB p65 (1:400; Thermofisher, Cat. No. PA1-186), mouse anti-HIF-1α(H1alpha-67) (1:80; Santa Cruz, Cat. No. sc-53546). After washing, cells were incubated with secondary antibodies for 45 min RT, washed, and mounted with 4′,6-diamidino-2-phenylindole (DAPI)-containing mounting solution (both from Sigma). Secondary antibodies used were: Alexa-Fluor 546-anti-mouse (1:300; Invitrogen), Alexa-Fluor 488-anti-rabbit (1:300; Invitrogen). Digital images were captured with a Zeiss Observer.Z1 microscope equipped with the Apotome.2 acquisition system (Zeiss, Oberkochen, Germany).

Cells were fixed using InsideFix Solution (Miltenyi Biotec, Bologna, Italy) and incubated overnight at 4 °C with primary antibodies diluted in InsidePerm solution (Miltenyi, Bologna, Italy). The antibodies used were rabbit anti-SIRT1(H300) (1:400; Santa Cruz Biotechnologies, Santa Cruz, CA, USA, Cat. No. sc-15404) and rabbit anti-acetyl-NF-kB p65 (Lys310) (1:30; Cell Signaling, Danvers, MA, USA, Cat. No. 3045). After washing, cells were incubated with secondary antibodies, diluted in InsidePerm solution, for 45 min RT. The secondary antibodies used were AF488-anti-mouse (1:300; Thermofisher Scientific, Waltham, MA, USA) and AF488-anti-rabbit (1:300; Thermofisher Scientific, Waltham, MA, USA). After washing, slides were mounted with 4′,6-diamidino-2-phenylindole (DAPI)-containing mounting solution (Sigma-Merck, Darmstadt, Germany). Digital images were captured with a Zeiss Observer.Z1 microscope equipped with the Apotome.2 acquisition system (Zeiss, Oberkochen, Germany). The number of immunopositive cells with nuclear SIRT1 was determined by cell counting in at least five randomly selected fields/well.