Ar1109

Based on our previous report (Fan et al., 2017 (link)), the pancreas was fixed in 4% paraformaldehyde and then a small part of the complete pancreas was cut. The pancreas tissues were soaked in 15% sucrose and 30% sucrose dehydrated overnight, respectively. The dehydrated tissues were embedded in OCT Compound (SAKURA, 4583) and frozen in −80°C. The tissues were subsequently sliced into 8 μm sections using freezing microtome (LEICA CM1850UN).
Sections were fixed with 4% paraformaldehyde for 20 min. Then permeabilized with 0.5% Triton X-100/SDS/PBS for 20 min, and blocked with 5%FBS/PBS/1% BSA for 3 h at room temperature. The slides were then stained with insulin (CST, 3014) and glucagon (Boster Biological Technology, BM1621) antibody overnight at 4°C. After washed, slides were dark incubated with FITC-conjugated goat anti-rabbit IgG (ABclonal, AS011) and Cy3-conjugated goat anti-rabbit IgG (ABclonal, AS008) for 2 h at room temperature. Sections were then washed with PBST and PBS for 5 min, followed incubated with Hoechest 33342 for 10 min at room temperature. After three washes with PBS, the slides were covered with fluorescence decay resistant medium (Boster, AR1109). Pancreatic sections were imaged using a light microscopy (Olympus BX51). The insulin-positive area and pancreatic area were measured using ImageJ software (Rockville, MD, United States).

Sperm suspensions from 5 mice of each group were diluted in distilled water, smeared on a cover glass, and dehydrated at room temperature for 2 h. Sperm deagglutination solution (25 mM DTT, 0.2% Triton X-100 and 200 IU/mL heparin) was added dropwise to the smear and incubated in a 37°C incubator for 15 min. The sperm deagglutination solution was discarded, and the 3.7% paraformaldehyde solution was used for fixation for 20 min. After washing with PBS and blocking with 5% BSA for 2 h at room temperature, the sample was incubated with rabbit-derived anti-H3K27me3 (A2363, ABclonal) primary antibody for 2 h. After washing three times with PBS, the goat anti-rabbit FITC-labeled secondary antibody (AS011, ABclonal) was added and incubated for 1 h. Hoechst 33342 (14533, Sigma) was used to stain the sperm nucleus. Finally, the sample was mounted with antifluorescence quenching mounting fluid (AR1109, BOSTER) and observed under the oil microscope of Olympus IX-53 with the microscopic image acquisition system (CellSens Dimension) (Olympus, Tokyo, Japan). Five sections were obtained from each mouse. Five high-power fields (×1000) were randomly selected, and ImageJ software (NIH) was used to analyze the average fluorescence intensity value of H3K27me3 in sperm.

H9C2 cells growing on the glass slide were fixed with 4% polyformaldehyde at room temperature for 20 min, washed with precooled PBS 3 times for 5 min, added to 0.01% Triton X-100, well ventilated at room temperature for 10 min, washed with PBS 3 times for 5 min, sealed with PBST containing 1% BSA at room temperature for 1 h, and incubated with 500 μL diluted antibodies GRP78 (ab21685; Abcam) and caspase3 (ab179517; Abcam) at 4°C overnight. On the following day, the cells were washed with PBST 3 times for 5 min, incubated with 1:200 diluted HRP-labelled antibody in a wet box for 1 h, and washed with PBST 3 times for 5 min. After adding a drop of DAPI (AR1176; Boster) to the center of the glass slide, the cells were incubated for 5‒10 min away from light and washed with PBST 3 times for 5 min. After dripping a drop of anti-fluorescence attenuation sealing agent (AR1109; Boster), images were captured under a fluorescence microscope (Nikon).

Following fixation with 4% paraformaldehyde, mouse pancreatic tissue sections were processed for embedding in paraffin, and then deparaffinized utilizing xylene and ethanol. The sections were treated with Citrate buffer (C1010, Solarbio, Beijing, China) which was heated and subsequently immersed. The mixture was then reduced to low heat and left to process for a duration of 15 min. To prevent nonspecific binding, the sections were blocked with a solution containing 3% hydrogen peroxide for a duration of 20 min. Sections were incubated with either insulin or glucagon primary antibodies overnight at 4 °C, then incubated with secondary antibodies for 1 h. Cell nuclei were stained with a DAPI solution, and sections were blocked with an anti-fluorescence quenching sealer (AR1109, Boster, Wuhan, China). Lastly, the sections were captured using an inverted fluorescence microscope (NIB900, Leica Microsystems, Germany).

Double immunofluorescence labeling was used to determine the colocalization of GFAP and GSNO in the colon. After placement in 4% paraformaldehyde, the samples were sliced, immersed in 0.01 M citrate buffer (pH 6.0), heated until boiling, washed with PBS, and blocked with 10% serum (Zhejiang Tianhang Biotechnology, China) for 30 min. Primary antibodies [anti-GFAP, rabbit monoclonal antibody, 1:100 (Bioss, China); anti-GSONR, mouse monoclonal antibody, 1:100 (Proteintech, United States)] were added to the samples. After the samples were washed with PBS, a secondary antibody [FITC-labeled goat anti-rabbit IgG (ServiceBio, China); Cy3-labeled goat anti-mouse IgG (ServiceBio, China)] and 4′,6-diamidino-2-phenylindole were added and the samples were incubated at room temperature for 10 min, washed with PBS, sealed with fluorescence decay-resistant sealing tablets (AR1109, Boster, China), and examined under a fluorescence scanning microscope camera system (P250 Flash, Tangier, China).

The cell suspensions were collected separately into centrifuge tubes, and cells were pelleted by centrifugation at 800g for 3 min. Then cells were washed twice in PBS, smeared onto glass slides, air-dried, and fixed in 3.7% formaldehyde at 37°C for 15 min, followed by 0.1% Triton X-100 treatment at room temperature for 10 min. Then the slides were blocked with Goat serum for 1 h at room temperature. After that, cells were incubated with the antibody against NEK2 (1:50, sc-55601, Santa Cruz) and PKM2 (1:100, 4053, Cell Signaling Technology) overnight at 4°C. Then cells were incubated with Alexa Fluor 488 goat anti-rabbit lgG (A11008, Life Technologies) and Alexa Fluor 594 goat anti-mouse lgG (A11005, Life Technologies) for 1h at room temperature and washed with PBS. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) for 5 min, protected from light. Finally, the sections were sealed by an anti-fluorescence quencher (AR1109, Boster). Olympus FV1000 laser scanning confocal microscopy (Olympus, Japan) was used to obtain images.