Microscopy and imaging system

Immunohistochemistry was conducted to localize the expression of genes of interest in UVJ tissue samples. The UVJ tissues fixed in PFA were dehydrated, embedded with wax, and processed sections with 5 μm thickness. The sections were gone through dewaxing with xylene, antigen retrieval with sodium citrate buffer (pH 6.0 ± 0.1), subsequently incubated with specific primary antibodies (GNAQ, rabbit, Thermo Fisher, PA5-79318, 1:2000; ST6GAL1, mouse, Abcam, ab77676, 1:200, UK; proliferating cell nuclear antigen [PCNA], rabbit, Servicebio, GB11010, 1:2000, China; adipocyte differentiation-related protein [ADFP], rabbit, Huabio, ET1704-17, 1:1000, China) overnight at 4°C. The sections were then incubated with secondary antibody (Proteintech, China) for 1 h at room temperature and stained by using DAB (1:50) followed with hematoxylin counterstain. The stained sections were sealed and recorded as images with microscopy and imaging system (Olympus, Japan). The experiments were performed at least twice in 2 individuals each group per antibody staining.

The UVJ tissues were embedded in OCT and cut into 10 μm frozen sections in accordance with the standard procedures. The sections were stained with Oil Red O to detect lipids in UVJ tissues. Sequentially, the sections were processed into drying, 4% PFA fixation, dehydration with absolute propylene glycol, staining in prewarmed Oil Red O solution, differentiation in 85% propylene glycol solution, rinsing, and counterstaining in hematoxylin solution. The sections were photographed under microscopy and imaging system (Olympus, Japan).

The UVJ tissue samples fixed in PFA or embedded in OCT were processed for wax section or frozen section, respectively, in accordance with the standard procedures. The sections were stained with hematoxylin and eosin (H&E) to view the potential differences of UVJ tissues between high and low SS capacity individual egg layer. The sections were examined under microscopy and imaging system (Olympus, Japan). The number of SST was calculated per mm² by ImageJ and graphed using software GraphPad Prism 7.0. The statistical analysis was performed using the Student t-test (n = 7).