Hochest 33342

For live cells immunofluorescence, CD45⁺F4/80⁺CD206⁻ M1-like and CD45⁺F4/80⁺CD206⁺ M2-like macrophages sorted from GD6 mice uterine by FCM were incubated with anti-CYP26A1 antibody (1:50, Invitrogen, PA5-24602) for 1 h at 4°C. After washing, cells were incubated with fluorochrome-labeled secondary antibodies (1:200, Jackson ImmunoResearch Laboratories, 711-545-152) for 30 min, washed with PBS for twice times. Cells were then incubated with Hochest 33342 (10 μg/mL, Beyotime, c1022) at 37°C for 25 min. After washing, cell suspensions were dropped on glass slide and covered with cover glass for taking pictures. Images were acquired using Carl Zeiss LSM880 confocal microscope.

Apoptosis was measured by using a TdT-mediated dUTP-biotin nick end labeling (TUNEL) BrightRed apoptosis detection kit (Vazyme Biotech Co., Ltd, Nanjing, China). Briefly, after anthocyanin and/or Rosup treatments as stated early, the medium were removed and GCs were collected and washed in PBS twice, followed by fixation using 4% paraformaldehyde (4% PFA). Then the cells were plated on the pretreated glass slides and dried on a 37°C heating block. Then the slides were washed in PBS three times (5 min each), and incubated with 100 μL proteinase K solution (20 μg/mL) at room temperature for 5 min, and then washed with PBS three times. Then the slides were incubated with 100 μl 1× equilibration buffer sample at room temperature for 30 min, followed by an incubation in a moist chamber with 50 μL terminal deoxynucleotidyl transferase (TdT) incubation buffer (containing 34 μL ddH₂O, 10 μL 5× equilibration buffer, 5 μL BrightRed labeling mix and 1 μL recombinant TdT enzyme) in dark at 37°C for 60 min. Slides were washed with PBS containing 0.1% Triton X-100 and 5 mg/mL BSA three times and then stained with 10 μg/mL Hochest33342 (Beyotime, Nantong, China) for 5 min, and washed with fresh water three times. Cell samples were examined using a fluorescence microscope equipped with a filter set (620 nm).

Treated cells were plated on cell slides (WHB Scientific, Shanghai, China) in advance, then fixed by 4% paraformaldehyde (Servicebio, Wuhan, China) at room temperature, followed by cell permeabilization with 0.5% Triton X-100 (Sangon Biotech, Shanghai, China) and cell blockage with 10% goat serum (Servicebio, Wuhan, China). Then, the cell slides were immersed in primary antibody diluted buffer (MBOP, 1:200) in the dark at 4 °C overnight. On the second day, the diluted buffer was washed off, followed by incubation with secondary antibody Alexa Fluor 555-labeled Donkey anti-Rabbit IgG (H+L) (1:1000, Multi-Sciences-A0453) diluted buffer in the dark at room temperature for 1.5 h. Nuclei were counterstained by Hochest 33342 (Beyotime, 1:1000). Immunofluorescence images were captured through OLYMPUS IX83-FV3000 (Olympus, Tokyo, Japan).

Cysteine, citric acid, PEG200, ε-caprolactone, pyrene, 1, 3-disphenylisobenzofuran (DPBF), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and benzylalcohol were all obtained from Aladdin (Shanghai, China). Enzyme 435, 4-nitrophenyl chloroformate (NPC), cystamine dihydrochloride, and glutathione were provided by J&K Scientific LTD (Beijing, China). Doxorubicin hydrochloride (DOX·HCl) was supplied by Dalian Meilun biotechnology Co., LTD (Dalian, China). Biotin-PEG2000-NH₂ (Mw = 2000 Da) was purchased from Shanghai Ponsure Biotechnology Co. Ltd (Shanghai, China). 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA), Annexin V-FITC, propidium iodide (PI), and Hochest 33342 were purchased from Beyotime biotechnology Co., LTD (Shanghai, China). Cypate was synthesized referring to previous work. All other chemicals were obtained from Beijing Chemical Works (Beijing, China) and were used without further purification.

The qualified oocytes were transferred into TCM199 (Gibco, Brooklyn, NY, USA) with 2% (v:v) FBS. Mature oocytes were incubated in TCM199 medium (Gibco, Brooklyn, NY, USA) supplemented 5 μg/mL CB (Sigma-Aldrich, St. Louis, MO, USA; C6762) and 5 μg/mL Hochest 33,342 (Beyotime, Tongzhou, Beijing, China, C1022) about 5–10 min. Then, with the help of a micromanipulator, mature oocytes were enucleated and then rhBChE-positive GFFs were injected into the enucleated oocytes. After 90 min, these oocytes were performed electric fusion. Firstly, injected oocytes were transferred into a fusion solution for 3–5 min. Then, oocytes were placed into a fusion tank covered with the fusion solution.
The polar body of the oocyte was oriented parallel to the electrode and a DC pulse was applied 2 direct current pulses of 2.0 kV/cm for 25 μs using an ECM2001 Electrocell Manipulator (BTX Inc., San Diego, CA, USA). The fused embryos were transferred into TCM199 medium including 10 μg/mL of actinomycetes CHX (Sigma-Aldrich, St. Louis, MO, USA, C7698) and 5 μg/mL of CB for 4–5 h. Finally, the embryos were incubated to the developmental solution and transplanted the next day.
Sixty days after the embryo transfer, we would assess the pregnancy rate of the female goats via ultrasonography.

For detection of IL-23R, cells were cultured with 1:100 diluted goat anti-IL-23R (Abcam) at 4° C and then with 1:100 diluted rabbit anti-goat PE-conjugated secondary antibody (ProteinTech Group, Wuhan, China) at 4° C in the dark. After that, half cells were analyzed by flow cytometer and half cells were further stained with hochest33342 (5 μg/ml, Beyotime) for 10min followed by observation under the fluorescence microscope (LSM 710 and ConfoCor 3, Zeiss, Germany).
For detection of CD133, saponin permeabilized cells were incubated with PE labeled anti-CD133 (Miltenyi Biotec, Germany) and then were analyzed by FACS Calibur.