Animal sample preparation
A total of 10 geese eggs (5
Anser anser and 5
Anser cygnoides) were selected and the universities incubation procedure was followed. This study was conducted at the Jilin Agricultural University breeding farm in Jilin Province, Northeast China in 2019. The geese were reared under a semi-intensive deep litter system and released to an open area extended to their swimming pool. They were fed ad libitum and allowed free access to water and occasionally supplemented with green grass, the feeding method was according to the technical regulations of breeding geese and commercial geese (Approval number: DB22/T 2022-20140). Geese were exposed to constant natural photoperiod and room temperature. Six different periods were selected, 18; 35; 48; 68; 88; and 108 days for both
Anser anser and
Anser cygnodies (weight difference within 500 g). Dorsal skin in about 1/ cm
2 piece without feather shafts and subcutaneous fat was collected and sluiced in phosphate-buffered saline (PBS). Samples were frozen with liquid nitrogen and stored at -80
oC designed for different junctures, while the other residual samples were secured in 10% phosphate-buffered formalin for tissue sectioning.
Primer design and synthesis
Two primers were used to detect the expression of the FZD4 gene in goose feather follicles. The primers were designed from the nucleic acid sequence of
Anser cygnoides domesticus (NCBI Reference Sequence: XM_01319 9825.1), with b-actin as an interior control. All the used primers were commercially designed and synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China) and were purified by polyacrylamide gel electrophoresis (Table 1).
Total RNA extraction and complementary DNA (cDNA) synthesis
Total RNA was extracted from the skin and feather follicle samples with RNAiso Plus (TaKaRa, Beijing, China) and cDNA was generated from total RNA after reverse transcription reaction. The total RNA was treated with DNase I (Ambion/Life Technologies) to remove the genomic DNA. The RNA quantity and concentration were measured with a spectrophotometer (Agilent Technologies, USA); the purity and degradation were also assessed on 1.0% agarose gels before proceeding to the next experimental procedures. Thereafter, the first-strand cDNA was synthesized using a cDNA Synthesis Kit (TOYOBO, Japan) according to the manufacturer’s instructions. cDNA was synthesized by reverse transcriptase using 2 µg of total RNA, 1 µL of RT enzyme mix, 1 µL of primer mix, 4 µL of 5XRT buffer and finally, Nuclease-Free Water added to a total volume of 20 µL; the reaction conditions of the reverse transcription reaction were used: 37
oC, 15 min and the enzyme deactivation was 98
oC, 5 min. The final products were kept at 4
oC for daily experimental usage and -20
oC for longer storage.
Quantitative real-time PCR
Quantitative real-time PCR amplification reactions were carried out in a final volume of 20 µL comprising 16.4 µL Premix (includes 10 µL SYBR and 6.8 µL ddH
2O), 0.6 µL of each primer and 2 µL template cDNA. All sequences of primers were shown in Table 1. Amplification conditions were as follows: pre-denaturation at 95
oC for 5 min, 45 cycles of amplification (95
oC for 15 s and 59
oC for 60 s). A melt curve analysis was performed from 60
oC to 95
oC by taking readings every 0.3
oC. The relative quantification of gene expression was detected in triplicate per sample. The 2
-ΔΔCT method was used for analyzing gene expression levels and using b-actin as an internal control. The PCR reaction was completed on a Bio-Rad CFX Real-time PCR Detection System and software (BIO-RAD, California, USA) used to assess the mRNA quantitative expression.
Western blot analysis
Different samples (about 30 mg) were homogenized in 200 μL RIPA buffer (10 μL of phosphatase inhibitor, 1 μL of protease inhibitor and 5 μL of 100 mM PMSF per 1 mL) followed by centrifugation at 12000 rpm, at 4oC for 15 min until the middle natant was separated into a clear and whole protein extract. The protein concentrations were determined using the BCA protein concentration assay kit (Sangon biological, C503021-0500). Thereafter, 20 μg total protein was pipetted onto a 10% SDS-PAGE Gel Electrophoresis Kit (Beijing liuyi instrument factory, DYY-6C) for each sample and initially set at 90 V and the voltage was increased to 160 V for 90 min when the protein samples enter the separation gel. The electroblotting was carried out in a transfer liquid at 300 mA for 59 min.
The membranes for FZD4 detection were blocked and incubated with 5% skim milk powder for 1.5 to 2 h at 25oC room temperature, followed by washing membranes with TBST 3 times for 5 min then incubating overnight at 4
oC immersed with FZD4 primary antibody (1:1000, LS-C201393, LSBio) and b-actin antibody (Biosynthesis Biotechnology, Beijing, China). The membranes were rinsed with 1_PBST (phosphate-buffered saline with 0.1% Tween-20), then incubated with an HRP Affinipure conjugated Goat-Anti-Rabbit IgG(H+L) secondary antibody (Wuhan, Hubei, China) for 1h at room temperature. Membranes were washed three times with 1_PBST at room temperature for 15 min. ECL test kit (Millipore WBKLS0100, Germany) was used to envisage membranes. Chemiluminescence protein bands were quantified by ImageJ software and protein levels were normalized by b-actin as an internal control.
Immunohistochemical
The different post-natal skin samples were fixed for 48 h and the condition was as follows: pH 7.4, at 4
oC in 4% paraformaldehyde. The samples were immersed in different concentrations of ethanol (75%, 85%, 90% and 95%) and immersed in 100% xylene twice for 10 min each time. Treated tissues were embedded in paraffin and 5ìm-thick sections were cut and then stretched and baked at 65
oC for 3 h and soaked twice in xylenes. The treated samples were washed with PBS (3 × 5 min). The slides were placed in citrate buffer solution (pH 6) for 2 × 15 min to antigen repair by heating. Tissue sections were washed with PBS (3 × 5 min). Endogenous peroxidase activity was inactivated by immersing slides in 3% H
2O
2 at room temperature for 30 min. The slides were washed with PBS (3 × 5 min). Subsequently, slides were blocked for 20 min with 3% goat serum at room temperature after deparaffinization, rehydration and enzyme digestion. And incubated with 50 ìL of the IHC-plus polyclonal rabbit anti-human FZD4 antibody c-terminal at 4
oC overnight (LSBio) and the sections subjected to the incubation were rewarmed for 45 min. After washing with PBS (3 × 2 min), the slides were incubated at room temperature for 60 min with 20 μL secondary antibodies. Hereafter, the tissues were washed thoroughly with PBS (3 × 5 min) and followed by DAB staining (5 to 10 min).
The stained sections were rinsed with tap water for 10 min then counterstained with hematoxylin for 2 min. The sections were decolorized in 1% hydrochloric acid-ethanol for 10 to 15 s, placed in distilled water and processed by PBST at about pH 7.4 for 5 to 10 min. The stained sections were rinsed with tap water for 15 min, then dehydrated and translucent by different concentrations of ethanol and xylene. Slides were mounted and sealed with neutral resin. The slides were examined under a fluorescence microscope (Olympus, Tokyo) and conventional light microscopy (Olympus, Tokyo).
