Anti atp5a antibody

In total, 2.5 μm sections of TMAs and paraffinized cell pellets were placed on glass slides and dried for 10 min at 60 °C, dewaxed for 10 min in HistoChoice Clearing Agent (Sigma, H2779), and rehydrated by consecutive washes in ethanol: 100%, 95%, 70%, and 40% v/v for 30 sec each. Heat-induced antigen retrieval was performed at 95 °C for 40 min with Dako Target Retrieval Solution of pH 9 (Dako, S2367). After cooling at room temperature, the slides were incubated 2.5% BSA (Fluka Analytical, 05488) in TBST 0.1% for 1 hour at room temperature. Anti-ADPr [31 (link), 32 ] (rabbit, 1:500 dilution) and anti-ATP5a antibody (Abcam, mouse, 1:250) diluted in the blocking solution were incubated overnight at 4 °C. Secondary antibody Alexa Fluor 488 AffiniPure goat anti-mouse (Jackson ImmunoResearch, 115–545–003, 1:500 dilution) and Cy3 AffiniPure goat anti-rabbit IgG (Jackson ImmunoResearch, 111–165–144, 1:250 dilution) were incubated for 2 hours at room temperature. Nuclear counterstaining was performed DAPI (Biolegend, 422801, 1:10,000 dilution). Microscope cover glasses (Menzel–Glaser, 7001023) were mounted with Mowiol mounting medium.

Male flies were anesthetized, and the indirect flight muscle was dissected in cold SDM. The muscles were then fixed for 30 min with 4% paraformaldehyde at room temperature. After fixation, samples were washed three times with 0.1% PBST (0.1% triton in 1× PBS) for 5 min each time. The samples were blocked with 1% normal goat serum (NGS)/0.1% PBST for 30 min at room temperature. The samples were then incubated with anti‐ATP5A antibody (1 : 250; Abcam) in 1% NGS/0.1% PBST overnight at 4 °C. The next day, the samples were washed three times for 5 min in 0.1% PBST and then incubated in 1% NGS with 0.1% PBST containing Alexa Fluor 488 anti‐mouse IgG (Thermo Fisher) at room temperature for 3 h. Subsequent to this, the samples were washed three times in 0.1% PBST and mounted in VectaShield mounting medium (Vector Laboratories, Burlingame, CA, USA). Images were captured using a laser confocal microscope (Zeiss LSM 710) and analyzed with ImageJ [18 (link)].