Fluoromount g medium

Fluorescent in situ hybridization (FISH) experiments were performed on 4 mice per group to confirm the lack of Malat1 in cell nuclei of Malat^−/− mice and to qualitatively evaluate the effect of hypoxia on Malat1 expression in Malat^+/+ mice. Mice were anesthetized and then perfused through the heart with sterile saline solution until the liver was translucent. The lung was subsequently excised and fixed by perfusing formalin (10%, containing 4% paraformaldehyde) for 5 min. The fixed lung was embedded with OCT and cut in 10 μm-thick sections. Stellaris® FISH probes, consisting of a set of mixed oligonucleotides recognizing mouse Malat1 (NCBI gene ID:72289, NR_002847.2, from nucleotides 751 to 6982) and labelled with QASAR 570 Dye (SMF-3008-1, Biosearch technologies, Inc., Petaluma, CA, United States), were hybridized as recommended by the manufacturer onto at least two lung sections per mouse. Nuclei were stained with Hoescht (Sigma-Aldrich, ON, Canada) in Fluoromount G medium (Electron Microscopy Sciences, Hatfield, PA, United States). Imaging was performed on a Zeiss Observer Z1 (objective 63X with oil) coupled with LSM 800 lasers system (Zeiss, ON, Canada) and analyzed with Zen Blue software (Zeiss, ON, Canada). Fiji ImageJ software was used to quantify RNA FISH signals on a total of 3–5 pictures per animal.

Cells were fixed in 4% paraformaldehyde for 10 min, incubated in 100 mmol/L glycine for 10 min, permeabilized with 0.5% Triton X-100 for 5 min, and blocked with 1% BSA for 30 min. Primary antibodies (PARP1 and PAR referenced in the above section) were incubated for 2 h at room temperature and secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 555 (CST) were incubated for 1 h. Nuclei were counterstained with DAPI and slides were mounted in Fluoromount-G medium (Electron Microscopy Sciences). Microscope images were acquired using an LSM 710 confocal microscope (Carl Zeiss) mounted on an Axio Observer Z1 microscope (Carl Zeiss) equipped with a Plan-Apochromat X63/1.4 NA oil-immersion objective. Image acquisition and analysis were performed using LSM ZEN software (Carl Zeiss).