Animals
The experimental yaks were divided into four groups: 1-day old, 30-days-old, 180-days-old and adult. Three yaks were selected for each group, regardless of sex, and purchased from a local herdsmen in Haiyan County of Qinghai Province. All of the yaks showed a good nutritional status, and appeared healthy with no apparent diseases or conditions. The yaks were sacrificed by exsanguination in a slaughterhouse. The lungs were obtained immediately after the yak had died, and tissue samples were immediately collected from the diaphragmatic lobe of right lungs (to ensure that obvious blood vessels and the trachea were not gathered). The tissue samples were divided into three parts. One part was cut into 1 cm3 sections and fixed with 4% paraformaldehyde (PFA). The other two parts were cut into 1 mm3 pieces; one part was fixed with 2.5% glutaraldehyde, and the other was put into a freezing tube and placed into liquid nitrogen.
Ethics statement
This study was approved by the Institutional Animal Care and Use Committee of Qinghai University (Xining, China). All methods were carried out in accordance with the ARRIVE guidelines and the Animal Ethics Procedures and Guidelines of the People’s Republic of China. No local regulations or laws were overlooked. All yaks used in this study were purchased from local farmers.
Haematoxylin and eosin staining
Lung tissue samples (1 cm3) were fixed in 4% PFA, dehydrated in 30%, 50%, 75%, 95% and 100% ethanol and then treated with xylene before embedding in paraffin. Paraffin-embedded lung tissues were cut into 4 µm sections. The sections were deparaffinized in xylene, and sections were stained either with haematoxylin and eosin (HE) (Y&K Bio, Xi’an, China) or Masson’s trichrome stain, to examine general morphology.
Immunohistochemistry
The unstained, deparaffinized sections were rinsed with Phosphate Buffered Saline with Twen-20 (PBST) 3 times for 5 min each time. Then, endogenous peroxidase was quenched using 3% peroxide-methanol at room temperature in the dark for 25 min, and then the samples were placed on a decolorizing shaking table 3 times, for 5 min each. The slides were then incubated with 3% foetal bovine serum (Sangon Biotech, Shanghai, China) at room temperature for 25 min. The serum was discarded, and rabbit anti-cattle CD34 and rabbit anti-CD34 polyclonal antibodies (Proteintech group, Wuhan, China) diluted in phosphate buffer saline (PBS) were added. CD34 is a transmembrane glycoprotein known as an angiogenesis marker. The sections were incubated in the primary antibodies overnight at 4 °C. Then, the sections were rinsed in Phosphate Buffered Saline with Twen-20 (PBST) (3 × 5 min), goat anti-rabbit IgG was added, and the sections were incubated for 30 min at 37 °C. 3,3-Diaminobenzidin (DAB) was added to the sections to visualise antibody binding, and the sections were washed 3 times in PBST. Haematoxylin was used to counterstain the nucleus prior to the samples being dehydrated and mounted.
An Olympus BX51 microscope was used to take photomicrographs of the microstructures, images depict 1000× magnification. Transmission electron microscopy.
The TEM lung tissue samples were processed using previously published methods16. Fresh lung samples (1 mm3) were fixed with glutaraldehyde (2.5%, 24 h) and postfixed with osmium tetroxide (1%, 2 h). The samples were dehydrated in a series of increasing concentrations of ethanol and embedded in Epon812. After preparing semithin sections, ultrathin sections were double stained with uranyl acetate and lead citrate. A 10,000× magnification was used to observe and photograph the sections with a JEM 1230 electron microscope (JEOL, Tokyo, Japan) set at 120 kV.
Quantitative real-time PCR (qPCR)
The gene expression levels in lung tissues from the yaks in the four age groups were analysed using qPCR. Total RNA was isolated with TRIzol® reagent (Invitrogen, CA, USA). cDNA was obtained by reverse transcription of total RNA using the SYBR PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time; Takara, Dalian, China). The forward and reverse primers sequences for the qPCR are shown in Table 1. The genes expression levels were detected using TB Green™ Premix Ex Taq™ II (TIi RNaseH Plus; Takara, Dalian, China) according to the manufacturer’s instructions. The 2−ΔΔCT method was used to analyse the relative expression of target genes, and the housekeeping gene β-actin was used for normalization.
Western blot analysis
Equal amounts of proteins of yak lung tissue in different development stages were harvested. These proteins were separated on 10% polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Sangon Biotech, Shanghai, China). PVDF membranes were blocked in 10% non-fat (skimmed) milk for 3 h and then incubated in rabbit anti-VEGFA polyclonal antibody (OriGene, Maryland, USA) at 4 °C overnight. The membranes were then incubated with a goat anti-rabbit IgG antibody (Abcam, Cambridge, UK) for 2 h being washed 3 times (10 min / time) with Tris-buffered saline with Twen-20 (TBST; containing 0.1% Twen-20). All antibodies were diluted according to the manufacturer’s instructions. Immunoblots were analysed by autograph using a Gel Doc™ XR + Gel documentation system (BIO-RAD, California, USA).
Statistical analysis
The experimental data are showed as the mean ± standard deviation (SD). The differences between the four groups were compared using one-way ANOVA. P values at less than 0.05 were considered significantly different.
Source: Ecology - nature.com
