Fv10 laser scanning confocal microscope

The commercial FISH Kit purchased from Bersinbio (Guangzhou, China) was used for the FISH assays following the manufacturer’s procedures. Briefly, hBMECs were fixed in 4% paraformaldehyde for 30 min at room temperature and washed with PBS twice. Cells were incubated with Proteinase K solution for 15 min at 37°C, washed twice with PBS for 5 min, and then mixed with 1% fixation solution for 10 min at room temperature. Fixed samples were incubated with 70% ethanol for 5 min, 85% ethanol for 5 min, and 100% ethanol for 5 min at −20°C before drying. Samples were next incubated with pre-hybridization solution for 30 min at 37°C, followed by incubating in the hybridization buffer with specific probes for circ_2858 (5′-Cy3-TGCCACTGCCAACCTGTGACTTGTTT-3′) as well as miR-93-5p (5′-FAM-CTACCTGCACGAACAGCACTTTG-3′) for 18 h at 37°C. After strict wash in SSC buffer, 1 μg/mL of 4′,6-diamidino-2-phenylindole (DAPI, Beyotime Biotechnology, China) was used for nucleus staining. Fluorescent images were acquired with an Olympus FV10 laser scanning confocal microscope (Olympus, Tokyo, Japan).

Cells were seeded in Lab-Tek II Chamber (Nulg Nunc International, Rochester, NY, USA), fixed with 3.7% methanol-free formaldehyde for 10 min, permeabilized with 0.1% Triton X-100 for 5 min and incubated with 10% pre-immune goat serum in PBS for 45 min. Slides were stained with Bid-specific antibody (1 : 500) at 4 ^oC overnight and then with secondary goat anti-rabbit antibody conjugated with Alexa Fluor 488 (Life Technologies) in dark chamber for 1 h. For live-cell imaging, cells transfected with pEGFP-N1-Bid were grown on MatTek Chamber Slides (MatTek corporation, Ashland, MA, USA). In both approaches, cells were co-stained with MitoTracker Red CMXRos (Life Technologies) according to the manufacturer's instructions. Images were captured using Olympus FV10 laser scanning confocal microscope using identical confocal settings for all samples and processed using Olympus FV10 1.7a viewer (Olympus, Center Valley, CA, USA) and Photoshop CS2 (Adobe Systems, San Jose, CA, USA). Pearson's correlation coefficients were calculated using ImageJ software with the Just Another Co-localization Plugin.

The process was similar to IHC. The brain sections were incubated by the rabbit anti-FAM171A2 antibody together with the mouse anti-CD31 antibody and shaken overnight. After washing, sections were immersed into goat anti-rabbit Alex Fluor 488 and goat anti-mouse Alex Fluor 647 (FAM171A2 and CD31) or donkey anti-rabbit Alex Fluor 488 and donkey anti-goat Alex Fluor 594 (FAM171A2 and IBA1) for 2 hours, followed by washing and imaged using the Olympus FV10 laser scanning confocal microscope.