Methanol

PC12 cells were seeded at 4 × 10⁴ cells /mL and incubated for 24, 48, 72 and 96 h. Following incubation, the cells were fixed with 80% ice-cold methanol (Thermo Fisher Scientific, Waltham, MA, USA) for 5 min and the methanol was removed. After permeabilisation with 0.1% PBS/Tween for 20 min, cells were incubated in a blocking solution consisting of 10% Bovine Serum Albumin (BSA) with 0.3 M glycine (Sigma-Aldrich, St. Louis, MO, USA) with primary monoclonal antibodies for HIF-1α (ab16066) (Abcam, Cambridge, UK) at a concentration of 2 µg/1 × 10⁶ cells and incubated for 30 min at 22 °C. The primary antibody-blocking solution was then removed via centrifugation at 300× g for 3 min. The cells were washed with PBS, centrifuged for 3 min at 300× g and then incubated with 500 µL of secondary antibody DyLight 488 goat anti-mouse IgG (H + L) (Abcam, Cambridge, UK) (1/500 dilution) for 30 min at 22 °C. An FC500 flow cytometer (Beckman Coulter, Brea, CA, USA) was used to analyse the samples. At least 10,000 events were collected per sample. Data were analysed using Flowing Software (Turku Centre for Biotechnology, Turku, Finland).

Methanol and trifluoroacetic acid for the extraction of fractions were obtained from Fischer Scientific (Leicester, UK). For the pomace extraction, citric acid monohydrate was used as an acidifying agent, obtained from Penta Chemicals (Prague, Czech Republic). The carriers used in the microencapsulation process were maltodextrine 18–20 DE obtained from Astron Chemicals (Attica, Greece) and arabic gum powder from Nexira (Rouen, France). The materials used for the analysis of the samples were Folin Ciocalteu phenol reagent (2N) from Carlo Erba Reagents (Barcelona, Spain), sodium acetate 3-hydrate from Panreac Quimica SA (Barcelona, Spain), sodium carbonate anhydrous from Penta Chemicals (Prague, Czech Republic), sodium hydroxide from Panreac Quimica SA (Barcelona, Spain), 2,2-diphenyl-1-picryl hydrazyl (DPPH) from Sigma-Aldrich (Steinheim, Germany) and gallic acid (98% w/w) obtained from Acrōs Organics (Fair Lawn, NJ, USA). The standard compounds used were rutin trihydrate from Merck (Darmstadt, Germany), cyanidin galactoside from Extrasynthese S.A. (Genay Cedex, France) and chlorogenic acid from Fluka (Buchs, Switzerland). Water, acetonitrile and Methanol for HPLC analyses of the samples were obtained from Fischer Scientific (Leicester, UK).

Frozen tissues of CBC1 (100 mg) were individually ground with liquid nitrogen, and the ground tissues were resuspended in 500 μL of prechilled 80% methanol (Thermo, United States) and 0.1% formic acid (Thermo, United States) for vortexing. The samples were incubated on ice for 5 min and then centrifuged at 15000 rpm in a refrigerated centrifuge (ST16R, Thermo, United States) at 4 °C for 10 min. Some of the supernatant was diluted to obtain a solution containing 53% methanol (Thermo, United States) with LC-MS grade water. The samples were subsequently transferred to fresh Eppendorf tubes (500 μL) and then centrifuged at 15000×g and 4 °C for 20 min. Finally, the supernatants were injected into a HPLC-MS/MS system for analysis. In addition, equal volumes of the different experimental samples were taken and blended as quality control (QC) samples, which were used to evaluate the stability of the results of the whole experimental process and the correlation analysis.

Dechorionated embryos were transferred to a 1.5 mL microtube and mixed in 362.5 μL PBT (0.3% Triton X-100 in PBS), 12.5 μL 10x PBS, and 125 μL 16% formaldehyde, methanol-free (w/v) (Thermo Fisher Scientific, USA). Embryos in the 4% formaldehyde fixing solution (w/v) were shaken for 15 min at 200 rpm using a mini rotator/shaker (Thermo Scientific). Fixing solution was discarded, 500 μL heptane (Sigma-Aldrich, USA) and 500 μL methanol (Thermo Fisher Scientific, USA) were added, and samples were vigorously shaken by hand/vortex for 2 min. heptane, methanol, and embryos in the interphase were removed and discarded. Samples were washed 3 times with methanol before resuspension in 1 mL PBT containing 1 μL Hoechst 33,342 (20 mM) (Thermo Fisher Scientific, USA). After a 10 min incubation at RT, 2 × 1 min and 1 × 10 min washes with 1 mL PTB were carried out to remove excess dye. Embryos were then staged using the ECLIPSE Ts2 microscope (Nikon) based on Foe et al.,¹⁰ (link) and reference images for nuclear cycle divisions from others.³⁶ (link)^,37 Embryos in PBT were kept on ice during staging. Finally, PBT was removed, TRIzol was added to pooled embryos, and samples were stored at −80°C until RNA was isolated using a standard TRIzol RNA isolation protocol.

Ginsenoside Rg1 (purity > 98%) was purchased from Herbpurify CO., LTD (Chengdu, Sichuan Province, People’s Republic of China). DSS (molecular weight: 36-50 kDa) was bought from MP Biomedicals Inc. (Aurora, OH, USA). MS-grade formic acid was purchased from TCI (Shanghai, China) and methanol was purchased from Thermo (USA). Ultra-pure water was produced by a Milli-Q water purification system (Millipore, MA, USA). methanol (Purity > 99.0%) used was obtained from Thermo Fisher Scientific (USA). The reference substance of Trp and its metabolites used were as follows: Nicotinamide (Nam, Sinopharm Chemical Reagent Co., Ltd, China); Indole-3-formaldehyde (IAld, Yuanye, Shanghai, China); 3-Indolepropionic acid (IPA, Aladdin, Shanghai, China); Tryptophan (Trp, Sinopharm Chemical Reagent Co., Ltd, China); and 3-Indolyllactic acid (ILA, Aladdin, Shanghai, China).