Airyscan confocal microscope system

Cy3-labeled probes specific for circGLIS3 were designed and synthesized by GenePharma, and the signals were detected by a FISH Kit (GenePharma) according to the manufacturer’s instructions. FISH + IF was performed with a FISH in situ hybridization immunofluorescence counterstaining kit (C009, Gefan, Shanghai). Confocal images were captured using Zeiss AIM software and a Zeiss LSM 880 with Airyscan confocal microscope system (Carl Zeiss Jena, Oberkochen, Germany).

Mice were sacrificed by cervical dislocation and the brain was removed from the skull, briefly washed in pre-cold D-PBS, embedded in OCT and freezing in dry ice. Then, the fresh frozen brains were cryosectioned at 16μm on a Leica cryostat 3050 (Leica Systems). A DIG labeled ribo probe complementary to SLAMR lncRNA was prepared by in vitro transcription of cDNA templates by using SP6/T& RNA polymerases (DIG RNA labeling Kit, Sigma). Sense and Antisense strands of 250nt were prepared by PCR using mouse hippocampus cDNA as a template and transcript specific PCR primers and ligated to pCRII-TOPO Vector with dual promoters SP6 (for antisense strand) and T7 (sense strand).
After the sectioning the tissue was dried and acclimated at RT for 2 hours, washed with D-PBS fixed with a 4% PFA solution for 10 min at RT, washed again with D-PBS (3 times, 3 min each), wash with 0.2% glycine in D-PBS (5 min), D-PBS washes (2 x 5 min), acetylated in TEA solution for 10 min, pre-hybridized at 68°C for 1 hr and hybridize with the probes overnight (approx. 16 hrs). After hybridization the signal was visualized using the TSA Plus Fluorescein Systems from PerkinElmer (TSA plus Cyanine detection kit, Akoya) for DIG detection. Images were acquired by using Zeiss LSM 880 with Airyscan confocal microscope system.