Alexa fluor 488 labeled secondary antibody

Cells were seeded at a density of 5 × 10⁵ cells/mL; at around 60% confluency, cells were treated as indicated above. Cells were washed twice with PBS, fixed with (PERM/FIX buffer from BD) and cells were stained with anti-PRMT-5 (Abcam, Cambridge, UK), overnight at 4 °C. Cells were then washed with 1× PBS and reacted with the Alexafluor^®488-labeled secondary antibody (Abcam, Cambridge, UK) for 1 h at 37 °C; excess reagent was rinsed with 1× PBS. Genomic DNA was stained with 4′,6′-diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, CA, United States) according to manufacturer’s instructions. Slides were visualized by confocal microscopy using a Nikon Confocal Microscope (Nikon, Tokyo, Japan). PRMT5 subcellular localization (nuclear and cytoplasmic) were quantified by measuring fluorescence intensity profiles using Image J software, National Institutes of Health (NIH), USA (http://rsb.info.nih.gov/ij/index.html, accessed on 2 April 2021). A total of 30 cells per experiment were analyzed under 40× objective lens and all images were acquired using identical parameters.

Six, twelve or twenty-four hours after IM, rats were sacrificed by perfusion with NS through the ascending aorta, followed by injection of 4% paraformaldehyde. The brainstem was quickly collected and post-fixed for 24 h with the fixing solution at 4 °C, and cryo-protected by immersion in 30% sucrose. Coronal brainstem sections (30 μm thick) were collected. After rinsing in 0.1 M PBS (pH 7.4), sections were blocked with 5% normal donkey serum with 0.1% Triton X-100 for 1 h at 37 °C and incubated overnight at 4 °C with rabbit anti-c-fos primary antibodies(1:3000, abcam, USA). The sections were then incubated for 1 h with Alexa Fluor 488-labeled secondary antibody (1:1000, abcam, USA). Images were captured from NTS at 10 × magnification using a NIKON A1R laser confocal microscopy. At least 5 nonadjacent sections were used to for counting the c-fos expression in the NTS by an observer blind to the treatment.

For immunofluorescence, cells cultured on coverslips were fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 for 15 min and blocked with 3% BSA (all in PBS) for 30 min. Cells then were incubated sequentially at 4°C overnight with primary antibodies and then with Alexa Fluor 488-labeled secondary antibodies (Abcam). Nuclei were counterstained with DAPI (Vector Laboratories, 1:500). Confocal fluorescence images were taken with a Leica TCS SP8 confocal microscope.

The cells were plated on crystal 6-well slice at the density of 5 × 10⁵/ml in the culture plates, and treated with Dex and different groups of exosomes for 24 h. The samples were washed thrice in PBS, fixed in 4% paraformaldehyde, and permeabilized for 15 min with 0.1% TritonX-100 in PBS. The 5% bovine serum albumin was used to block cells at 37 °C for 1 h, followed by washing with PBS, and culturing with primary antibodies against Col-I (1:200, abcam, USA), or overnight at 4 °C. The TRITC Phalloidin, Alexa Fluor^®488-labeled secondary antibodies (1:400, abcam, USA) and DAPI solution (1:10) were added in sequence for 20 min, 1 h and 5 min at room temperature respectively, followed by 3 times rinsing in PBS. The images were captured by an inverted fluorescence microscope (Olympus, Japan). As for histofluorescence, the sections were deparaffinized and hydrated and incubated with a mixture of EMCN (1:200, Invitrogen, USA) and CD31(1:200, Invitrogen, USA). After washed with PBS, sections were incubated with secondary antibodies: Alexa Fluor 488-conjugated and Alexa Fluor 594-conjugated (1:200, Abcam) at room temperature for 30 min. The nuclei were stained with DAPI solution. The images were captured by an inverted fluorescence microscope (Olympus, Japan).

To verify the expression level of CD51, cells were seeded into glass-bottom dish with thickness of 0.16–0.19 mm (In vitro Scientific, USA). After entering exponential growth curve, the cells were fixed with 4% paraformaldehyde at room temperature for 15 min. After washing with PBS three times, cells were blocked in the 0.1% PBST supplemented with 10% horse serum for 30 min at room temperature. Then, the cells were incubated with mouse anti-CD51 primary antibody (Ancell, USA, 202–820, dilution ratio: 1:100) in blocking solution containing 4% bovine serum albumin and kept overnight at 4 ℃. Alexa Fluor^® 488-labeled secondary antibodies (Abcam, USA, ab150113, dilution ratio 1:500) were employed to visualize the expression and localization of CD51 protein under Zeiss LSM 780 confocal microscope with ×63 water immersion objective (NA 1.2). Additional images to visualize the expression and localization of CD51 protein of KYSE-30 and OE33 cells (both wild-type and knockout cells) were obtained using Leica DMi8 inverted microscope (Leica, Germany) with GFP filter (Ex: 450–490 nm; Em: 500–550 nm) and ×63 oil immersion objective (NA 1.4).