Pentobarbital sodium

Six-week male CD-1 (ICR) mice were purchased from Shanghai Laboratory Animals Center. For HSV-1 infection, mice were anesthetized by intraperitoneal injection of 0.4 ml of a mixture consist of 500 ug/ml xylazine hydrochloride (Sigma, X1251) plus 4 mg/ml pentobarbital sodium (Solarbio) in sterile saline. Then 2 x 10⁵ pfu of virus was added onto each scarified cornea in 3 μl of PBS. For TG acquisition, mice were euthanized by cervical dislocation while under anesthesia with isoflurane and TG were collected into the lysis buffer and placed on dry ice before RNA extraction and placed into -80°C as described previously [59 (link)]. For western blot analysis, we homogenized TG in lysis buffer (Solarbio, R0020) and extracted protein according to the manufacturer’s protocol. For determination of viral titers in TG, TG were homogenized in cell culture media for titer determination. For eye swab collection, mice were anesthetized in an induction chamber with isoflurane (3% in oxygen 0.5 ml/min) using a V1 Table Top anesthesia machine (Colonial Medical Supply). Both eyes of each mouse were swabbed with cotton-tipped applicators, suspended in 1 ml of cell culture medium.

All rats were anesthetized by intraperitoneal injection of 2% (w/v) pentobarbital sodium (40mg/Kg, Solarbio Science & Technology, Beijing, China). A McFarlane flap model whose procedure was modified based on the method reported by Kelly CP was established on rat dorsum (the same position in all rats) [18 (link)]. Laboratory rats were ventilated and fixed on a thermostatic operating table. Barium sulfide: talc: flour = 1: 1: 1 with water stirred into a paste depressant was applied to the rat dorsum. After the removal of the hair and conventional disinfection, rat dorsal as the longitudinal axis of the flap and connection of rat lliac crests as the pedical, the deep fascia shallow and flesh deep dissection between the anatomy were set off to establish a random skin flap (9cm×3cm). Immediately, flaps were sutured to the donor bed using a wedged-on cutting needle and continuous 4-0 silk sutures. The area around incision was disinfected by the poly-cup copper iodine and smeared with chlortetracycline ointment. The surgical procedures were implemented strictly by one experimental staff under aseptic conditions. For the interest of analysis, the flap area was divided into three equal sized zones (Figure 9) and marked as proximal (region I), intermediate (region II) and distal (region III).

On week 4 and 6 postoperation, three rats from each group were sacrificed under general anesthesia with intraperitoneal injection of 1% pentobarbital sodium (0.1 mL/100 g, Solarbio, Beijing, China). The femurs were harvested and processed following a previously published protocol [28 (link)]. Briefly, the isolated femurs were fixed in 10% neutral-bufferd formalin for 24 hrs and then immersed into 9% formic acid for decalcification. Thereafter, the specimens were dehydrated and embedded in paraffin. Several sections (5 μm) were cut along with the long axis of femour shaft and collected on glass slides for hematoxylin and eosin (H&E, Solarbio) and ALP staining (Solarbio) using standard protocols. After mounting with coverslips, the sections were observed and analyzed by an optical microscope.

Catalpol (98%) was purchased from Solarbio Science & Technology (Beijing, China). 3-Methyladenine was obtained from Solarbio Science & Technology (Beijing, China). The SIRT1 inhibitor EX527 was obtained from Sigma-Aldrich Chemical Company (Milwaukee, WI, USA). Diaminobenzidine (DAB) developer, pentobarbital sodium, and the H&E staining kit were obtained from Solarbio Science & Technology (Beijing, China). In the molecular studies, the following antibodies were used: anti-Cadherin 5 (A02632-2; Boster Biological Technology, Wuhan, China), anti-GAPDH (AP0063; Biogot Technology, Shanghai, China), anti-VEGF (A12303, ABclonal, China), anti-SIRT1, anti-LC3II, anti-Superoxide Dismutase 1 (SOD1), anti-Cathepsin D (CTSD), anti-Caspase 3 (CAPS3), and anti-Heme Oxygenase 1 (HO1) (13161-1-AP, 14600-1-AP, 10269-1-AP, 21327-1-AP, 19677-1-AP, and 27282-1-AP; Proteintech Group, Chicago, IL, USA); the primary antibody anti-Bax, anti-Bcl-2, anti-AMPK, anti-p-AMPK, anti-mTOR, anti-p-mTOR, anti-Cleaved-Caspase 3 (C-CAPS3), and antiendothelial nitric oxide synthase (eNOS) were obtained from Cell Signaling Technology (CST) (2772S, 15071S, 5832S, 2535S, 2983, 5536S, 9664S, and 32027S; Beverly, MA, USA); the primary antibody anti-SQSTM1/p62, anti-CD34 and anti-Matrix Metalloproteinase 9 (MMP9) were obtained from Abcam(ab56416, ab81289, ab283575; Cambridge, UK).

The high prolificacy sheep group (HP, n = 3, litter size = 3) and low prolificacy sheep group (LP, n = 3, litter size = 1) were selected from the nucleus herds of Hu sheep at Taizhou Sheep Industry according to their littering records (three consecutive lambing records) and polymorphism analysis of FecB [17 (link)], two groups of sheep in this study had similar numbers of dominant follicles and FecB genotype. All sheep were housed under the same conditions with free access to feed and water. Synchronous estrus of sheep was conducted according to previously described [18 (link)]. The estrous cycles of the ewes were adjusted by intravaginal progestagen sponges (30 mg; Ningbo Sansheng pharmaceutical Co., LTD, Zhejiang, China) for 11 days. Estrus was monitored by presentation of a buck fitted with an apron three times one day following sponge removal. The sheep were deeply anesthetized by intravenous administration of 3% pentobarbital sodium (30 mg/kg; Solarbio, P8410, China), and sacrificed by exsanguination in a healthy physiological stages at the second estrus (natural estrous), and endometrium was collected from the mid-part of uterine horns, and immediately frozen in liquid nitrogen for RNA extraction.

Limonin (C₃₂H₄₂O₁₄, purity ≥98%) was purchased from Solarbio (Beijing, China). Diaminobenzidine (DAB) developer, pentobarbital sodium, and the hematoxylin and eosin (H&E) staining kit were provided by Solarbio (Beijing, China). Rabbit anti–cadherin 5 was purchased from Boster Biological Technology (A02632–2). Rabbit anti-GAPDH was acquired from Biogot Technology (AP0063). Rabbit anti–vascular endothelial growth factor (VEGF), anti–superoxide dismutase 1 (SOD1), anti–matrix metalloproteinase 9 (MMP9), anti-HO-1, and anti-CAPS3 were acquired from Proteintech (19003-1, 10269-1, 10375-2, 21327-1, and 19677-1). Rabbit anti–endothelial nitric oxide synthase (eNOS), anti–cytochrome c (CYC), and anti-Bax were purchased from Cell Signaling Technology (12994, 14796, and 32027). Horseradish peroxidase (HRP)–conjugated immunoglobulin G (IgG) secondary antibody was purchased from Santa Cruz Biotechnology. Fluorescein isothiocyanate (FITC)–conjugated IgG secondary antibody was obtained from Boyun Biotechnology, and the 4′,6-diamidino- 2-phenylindole (DAPI) solution was purchased from Beyotime Biotechnology. The Electrochemiluminescence (ECL) Plus Reagent Kit was obtained from PerkinElmer Life Sciences and the BCA kit was acquired from TermoFisher Scientifc.