Paraffin wax

The intestinal samples were processed according to the method of Thompson et al. [14 (link)]. In brief, the intestinal samples were dehydrated with increasing concentrations of ethanol, cleared with xylene (Surgipath Medical Industries, Richmond, IL), and embedded in paraffin wax (Thermo Fisher Scientific, Kalamazoo, MC). Cross sections (5 μm) were stained with hematoxylin and eosin (GeneCopoeia, Rockville, MD). The stained sections were dehydrated with ethanol, cleared with xylene, and mounted with DPX mountant based on the method of Jiang et al. [15 (link)]. ImageJ software (National Institutes of Health, USA) was used to determine the morphometric measurements of villus height and crypt depth of the jejunum using an Olympus BX40 F-3 microscope (Olympus Cooperation, Tokyo, Japan) attached to a digital video camera (Q-imaging, 01-MBF-200R-CLR-12, SN: Q32316, Canada) [16 (link)].

Briefly, the intestinal samples were dehydrated with increasing concentrations of ethanol, cleared with xylene (Surgipath Medical Industries, Richmond, IL, USA), and embedded with paraffin wax (Thermo fisher scientific, Kalamazoo, MC, USA), and cut into 4-μm thick histological sections for hematoxylin and eosin staining. The tissue sections were measured under a microscope using a 40 × combined magnification, and an image processing and analysis system (Version 1, Leica Imaging Systems Ltd., Cambridge, UK). Villus height (VH); villus width (VW); crypt depth (CD); and VH/CD ratio (VH:CD) of the small intestine were determined by Program Image-pro Plus 6.0.

Ethyl acetate, chloroform, hexane, methanol and Xylene were purchased from Merck, Germany. DPPH (2,2-diphenyl-1-picrylhydrazyl), quercetin and streptomycin sulphate were from Sigma-Aldrich, Germany. Nutrient broth and Mueller-Hinton agar (MHA) were from Oxoid, United Kingdom. Diethyl ether was obtained from Emsure®, Germany. Formaldehyde was obtained from Scharlau, Spain. Paraffin wax was from Thermo scientific, United Kingdom. Hematoxylin was obtained from Fluka, Switzerland.

Intact ovaries collected from immature mice (3 weeks old) that were that were untreated controls or treated with FSH for 48 h were fixed in 4% paraformaldehyde (Boster, Wuhan, China) for 12 h at 4 °C. After washed with PBS and dehydrated, these tissues then were embedded in paraffin wax (Thermo, USA); and paraffin-embedded tissue sections (5 μm) were deparaffinized. After washing with PBS, the sections were treated with 10 mM citric acid buffer (pH 6.0) (Boster, Wuhan, China) for antigen activation for 15 min in boiling water. The sections were washed with PBS and blocked by 5% (v/v) BSA in PBS (Boster, Wuhan, China). Sections were incubated overnight at 4 °C with mouse monoclonal antibody specific to RBP4 at a 1:200 dilution in PBS (Proteintech, USA). In order to validate the specificity of RBP4 antibody, 5% nonimmune goat serum [26 (link)–29 (link)] or PBS [30 (link)] was used as negative controls instead of RBP4 antibody. After washed by 0.3% (v/v) Triton X-100 in PBS, sections were incubated with goat-anti-mouse second antibody (Boster, Wuhan, China) for 1 h at room temperature, stained using ABC kits, and counterstained with hematoxylin.

The histomorphologal parameters of the jejunal tissue samples were measured using previously published methods [34 (link), 38 (link)]. Briefly, the jejunal tissue samples were dehydrated and embedded with paraffin wax (Thermo fisher scientific, Kalamazoo, MC). The paraffin blocks were cut at 5-μm-thick cross sections using a microtome, then stained with hematoxylin and eosin (H&E) (GeneCopoeia, Rockville, MD), and examined under an Olympus BX40 F-3 microscope (Olympus Cooperation, Tokyo, Japan) attached to a digital video camera (Q- imaging, 01-MBF-200R-CLR-12, SN: Q32316, Canada) as described in Jiang et al. [34 (link)]. The morphometric measurements of villus height and crypt depth of the jejunum were analyzed by using the software of Image J (National Institutes of Health, USA).

Ovarian tissues were embedded in paraffin wax (ThermoFisher Scientific, Inc., Waltham, USA) after dehydration. Paraffin-embedded tissue sections (5 μm) were deparaffinized. The sections were blocked with the M.O.M kit (VECTOR) and then incubated with primary antibody (1 : 100 anti-PCNA antibody, Cell Signaling#2586S, 1 : 100 anti-DNMT1 antibody, GeneTex#GTX116011, 1 : 100 anti-TET2 antibody, Abcam#ab124297) in 0.3% (v/v) Triton X-100 in phosphate-buffered saline (PBS) (−) overnight at 4 °C. After washing with 0.3% (v/v) Triton X-100 in PBS (−), the ovaries were visualized with Alexa Fluor 568 goat anti-rabbit secondary antibody (1 : 100) (Sigma) or fluorescein isothiocyanate-conjugated goat anti-mouse IgG secondary antibody (1 : 100) (Sigma) and DAPI (Vector Laboratories, Inc., CA, USA). Digital images were captured using Keyence BZ-9000 microscope (Keyence, Co., Osaka, Japan).