Anti mtco2 antibody

The AON-treated ROs were fixed in 2% paraformaldehyde (Thermo Fisher Scientific) and 5% sucrose (Thermo Fisher Scientific) for 15 min at 4°C, followed by a 30-min incubation in 7.5% sucrose, 30 min in 15% sucrose, and 2 h incubation in 30% sucrose. The organoids were transferred to a cryomold and embedded in 7.5% gelatin (porcine skin; Merck KGaA) and 10% sucrose. The sample blocks were then frozen at −80°C. Sections of 10 μm thickness were sliced on a Cryotome FSE (Thermo Fisher Scientific), rehydrated in PBS, and stained following the protocol described by Cowan et al.⁴⁶ (link) ABCA4 was detected using the anti-ABCA4 3F4 clone (1:100; Abcam, Cambridge, UK), rhodopsin was stained using the anti-rhodopsin 4D2 clone (1:300, Invitrogen), mitochondria were detected with an anti-MTCO2 antibody (1:150; Abcam), and nuclei were stained with Hoechst 33,342 (1:1000; Thermo Fisher Scientific). Images were collected on an LSM 800 confocal microscope (Carl Zeiss, Oberkochen, Germany) using a 60× objective and analyzed with ZEN Blue edition (Carl Zeiss) using the maximum intensity projection.

To assess mitochondrial dynamics, human MDMs/HeLa cells were transfected with myc-tagged ORMDL3 plasmid or empty vector. Twenty-four hours post transfection, cells were fixed and stained. Mitochondria and ORMDL3 were stained with anti- MTCO2 antibody (Abcam), and anti- Myc antibody (proteintech) and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Acros Organics). Imaging was conducted with OLYMPUS BX61-FV1200-MPE microscope. ImageJ and Fiji (https://imagej.net/software/fiji/downloads) software were used to analyze mitochondrial length.

THP-1 cells were treated with TSLP for 2, 4, 6 or 8 h and analyzed by western blotting with anti-MTCO2 antibody (Abcam), anti-5′-AMP-activated protein kinase (anti-AMPK, Cell Signaling, Massachusetts, USA), anti-phospho-AMPK (Cell Signaling Technology, Danvers, MA, USA), anti-PINK1 (Cell Signaling Technology, Danvers, MA, USA), anti-LC3 (MBL, Nagoya, Japan), anti- phospho-Parkin (Ser65) Antibody #36,866 (Cell Signaling Technology, Danvers, MA, USA) and anti-GAPDH antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Western blotting analysis was performed as described in our previous work [46 (link)].