Dapi fluoromount g

Cells were cultured on 2-chamber-glass-slides (Electron Microscopy Sciences) for two days. Cells were then washed twice with PBS and fixed in freshly made 1% paraformaldehyde/PBS for 15 min at room temperature. After three PBS washes (5 min each), cells were permeabilized at 4°C for 15 min in 1% triton-X100/PBS. After three PBS washes (5 min each), slides were saturated in a 1% BSA/PBS solution for 15 min at room temperature and incubated with antibodies diluted at 1:200 in 1% BSA/PBS for 1h at room temperature. After three PBS washes (5 min each), cells were incubated with alexa-fluor 488 anti-mouse IgG or 568 anti-rabbit IgG secondary antibodies (Life Technologies) diluted at 1:500 in 1% BSA/PBS for 30 min at room temperature and in the dark. Finally, cells were washed in PBS three times and mounted in DAPI/fluoromount G (Electron Microscopy Sciences). After drying, slides were observed under a confocal microscope (Zeiss LSM700).

10-µm skin sections were washed twice with PBS, fixed with 4% PFA for 10 min and again washed twice with PBS-0.1% Tween. Subsequently, sections were incubated with ProtK (10 µg/ml) for 5 min and washed twice with PBS-0.1% Tween. For Tunel staining, the “In situ Cell Death Detection Kit-TMR red” (Roche) kit was used according to the manufacturer’s instructions. Briefly, 50 µl Enzyme solution and 550 µl Label solution were mixed and applied on the sections, incubated for 60 min at 37 °C in a humidified box in the dark. Slides were washed three times in PBS then mounted with DAPI. When co-stained for P-Cadherin, the ProtK step was omitted, and after the last three washes in PBS, slides were blocked for 2 h in 10% HISS in PBS. Sections were overnight incubated at 4 °C with Rat anti-Pcad antibody (1:100; BD, #MAB761), washed in PBS three times for 10 min, and incubated for 1 h at room temperature with Donkey anti-Rat Cy5 antibody (1:500; Jackson immunoresearch, #712-175-753). Sections were then washed in PBS three times for 10 min and mounted with DAPI FluoroMount-G (Electron microscopy sciences).

Dorsal skins were harvested, fixed in 4% PFA in PBS for 16 h at 4 °C, dehydrated in increasing sucrose concentrations (10%–15%–20%), embedded in Optimal Cutting Temperature (OCT) compound, frozen on liquid nitrogen, and cryosectioned (8–10 μm) (Cryostat Leica CM3050S). Sections were washed twice with PBS, fixed with 4% PFA for 10 min and again washed twice with PBST. Subsequently, sections were incubated with ProtK (10 µg/ml) for 5 min and washed twice with PBST. For Tunel staining, the “In situ Cell Death Detection Kit-TMR red” (Roche) kit was used according to the manufacturer’s instructions. Sections were washed 3 times in PBS and mounted with DAPI FluoroMount-G (Electron microscopy sciences BN17984-24).

Harvested tissues were fixed with 4% paraformaldehyde for 24 hours and then incubated with 30% sucrose for 72 hours. Tissues were then embedded in OCT (Sakura Tissue-Tek), sectioned (20 um) using a cryostat (Leica CM 3050 S), prepared on Fisherbrand™ Superfrost™ Plus microscope slides (Fisher Scientific, Canada) and stored at -80C. During immunostaining, frozen sections were washed with PBS, and incubated with 3% albumin bovine serum (ABS) dissolved in PBS containing 0.1% Triton X-100 for one hour to block unspecific binding. Cells were then incubated overnight with either anti-ICP4 antibody (1:200; Abcam, Cambridge, MA) or anti-ICP27 antibody (1:200; Abcam, Cambridge, MA) diluted in PBS containing 0.1% Triton X-100 solution at 4°C. The next day after washing 3 times with PBS, sections were incubated with goat anti-mouse IgG Alexa Fluor 488 secondary antibody (1:500; Invitrogen, Canada) for one hour at room temperature. After incubation with secondary antibody, sections were washed 3 times with PBS and mounted with Dapi Fluoromount G (Electron Microscopy Sciences). Sections were then visualized and imaged using a confocal microscope (Olympus, Canada).

WT and USP10-KO H4 cells were grown on glass coverslips coated with 5 μg/ml fibronectin for 24 h prior to experiments. Cells were incubated with 5 μg/ml puromycin for 2 or 3 h, then washed once with PBS, fixed in 4 % paraformaldehyde (PFA) in PBS for 20 min at room temperature, permeabilized with 0.1 % saponin (Sigma-Aldrich, 47036) for 20 min, and incubated with blocking buffer containing 0.2% BSA (Sigma-Aldrich, A7030). Cells were next stained with anti-SQSTM1 and anti-Ub primary antibodies diluted in 0.2 % BSA for 1 h at 37 °C, followed by staining with Alexa Fluor-conjugated secondary antibodies (Thermo Fisher Scientific) for 30 min at 37 °C. Cells were washed three times with PBS and once with distilled water and mounted with DAPI-Fluoromount-G (Electron Microscopy Sciences, 17984-24). Fluorescence was visualized on a Carl Zeiss LSM780 confocal microscope. Image analysis was performed with ImageJ.

Cells were plated onto poly-d-lysine–coated coverslips and transfected as described with indicated plasmids. At 24 h posttransfection, the cells were fixed with 4% (wt/vol) paraformaldehyde (Electron Microscopy Sciences, Cat#15710) and perforated with 0.1% (vol/vol) Triton X-100 in PBS, after blocking in 3% BSA in PBS. Cells were stained with primary antibody GFAP (Abcam, Cat #ab7260, 1:1,000), Na/K ATPase (Abcam, Cat #ab76020; 1:300) and then incubated with secondary antibodies anti-rabbit IgG (H+L) Alexa Fluor Plus 488 conjugated (Invitrogen, Cat #A11070; 1:1,000) and phalloidin-conjugated Alexa Fluor 568 (Invitrogen, Cat #A12380; 1:500), washed, and mounted onto slides with Dapi-Fluoromount-G (Electron Microscopy Sciences, Cat #17984-24). The fluorescence images were examined with a stimulated emission depletion microscopy (Leica TCS SP8 STED).