Lsm880 nlo 2 1 with big confocal microscope system

Cy3-labelled circNEIL3 probes and fluorescein amidite (FAM)-labelled miR-432-5p probes were designed and synthesized by RiboBio. A fluorescence in situ hybridization (FISH) kit (RiboBio) was used to detect the probe signals in CFPAC-1 and MiaPaca-2 cells according to the manufacturer’s instructions. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). All images were acquired with an LSM880 NLO (2 + 1 with BIG) confocal microscope system (Carl Zeiss).

For IF, mouse brain tissues obtained 24 h after MCAO were fixed in 4% of paraformaldehyde for > 48 h and transferred to a 30% sucrose solution for dehydration. Ten-millimeter-thick paraffin coronal brain slices were acquired and incubated with a primary antibody against PPARg (Cat#bsm-33436 M; Bioss, Beijing, China) and UBE2V2 (Cat#10,689–1-AP; Proteintech, USA) overnight at 4 °C, followed by incubation with donkey anti-mouse Alexa Fluor-conjugated secondary antibodies (1:200, Jackson ImmunoResearch) for 1 h at room temperature. Finally, all slices were washed in PBS three times and counterstained with 4′,6-diamidino-2-phenyl in- dole dihydrochloride (DAPI; Cat#0100–20; SouthernBiotech, USA) for 5 min. Images were captured using a fluorescence microscope (Olympus, Japan), and the numbers of PPARg-positive and UBE2V2-positive cells in the infarction border cortex were counted and analyzed using ImageJ software.
For FISH, the Cy3-labeled miRNA-193a-5p probe was designed and synthesized by Servicebio. The FISH kit (Servicebio, Beijing, China) was used to detect probe signals in mouse brain tissues co-stained with anti-UBE2V2 and anti-Ly6G antibodies, according to the manufacturer’s instructions. Nuclei were stained with DAPI. All images were obtained with an LSM880 NLO (2 + 1 with BIG) confocal microscope system (Carl Zeiss).