G1101 500ml

Liver tissues were fixed in 4% paraformaldehyde (Servicebio, G1101-500ML), embedded in paraffin, and sectioned at 5 μm. Paraffin-embedded liver sections were dewaxed, rehydrated, and stained with H&E, Masson’s stain, and Sirius Red, as described^{24 (link)}.

The distribution of HSF1 was assessed by immunofluorescence as described previously [26 (link)]. Briefly, the cells were fixed with 4% paraformaldehyde (G1101-500ML, Servicebio, Wuhan, China) for 15 min, permeabilized with 1% Triton X-100 (G5060-100ML, Servicebio, Wuhan, China) for 10 min, and incubated overnight at 4 °C with anti-HSF1 primary antibody (A13765, 1:100, Abclonal, Wuhan, China). Next, the cells were incubated with Cy3-labeled goat anti-rabbit antibody (BA1032, Boster, Wuhan, China) for 1 h and DAPI (AR1177, Boster, Wuhan, China) for 15 min at room temperature. The distribution of HSF1 was then observed by fluorescence microscopy (IX73-A12FL/PH, OLYMPUS, Japan).

After being fixed in 4% paraformaldehyde (G1101-500ML, Servicebio, Wuhan, China) fixative for 48 h, ovaries were washed, dehydrated, and then embedded in paraffin. Ovaries were cut into 5 micron slices. After H&E staining, the sections were sealed, and the ovarian tissue structure was photographed and observed under an optical microscope (Upright optical microscope, Eclipse E100, Nikon, Tokyo, Japan; Imaging system, DS-U3, Nikon, Tokyo, Japan). According to the classification criteria of growing follicles, antral follicles, and mature follicles, they are defined as follows: growing follicles have more than three layers of cubic granulosa cells and no antrum present [46 (link)]; antral follicles have multiple layers of granulosa cells and antrum present [1 (link)]; mature follicles are in the superficial layer of the ovarian cortex, and the follicular cavity is enlarged and filled with follicular fluid, which is round or oval in shape. The oocytes are located on one side of the follicle and form cumulus with the surrounding granulosa cells, and there are 3–4 layers of granulosa cells at the top of the cumulus [46 (link)]. The right ovaries of four individuals were used from each group and three sections of each ovary were examined. The average number of growing follicles and antral follicles per cross section were counted under the same magnification in each group.