Anti fading reagent

After 20 min of paraformaldehyde fixation, the samples were washed three times with PBS. The cells were then permeabilized for 10 min with 0.5% Triton X-100, washed three times with PBS, and blocked with goat serum for 1 h. Cellular samples were incubated with the indicated antibodies overnight at 4 °C, washed with PBS three times, and incubated with corresponding secondary antibodies for 1 h at 37 °C. Then, the samples were rinsed in PBS, counterstained with diamidino phenylindole (DAPI) (Beyotime, Shanghai, China, P0131), and mounted with anti-fading reagent (Solarbio, Shanghai, China). Representative images were visualized under a confocal microscope (Nikon A1R, Tokyo, Japan).

The cells were pre-seeded on glass slides. After culture for certain times, the cells were fixed with 4% paraformaldehyde for 15 min, permeated with 0.1% TritonX-100 (Beyotime) for 30 min, and blocked with goat serum for 10 min at room temperature. Subsequently, the cells were incubated with antibody against FBP1 (1:100; cat. no. 12842–1-AP, Proteintech) or CK19 (1:50; cat. no. A0247, Abclonal) at 4 °C overnight. After washing with PBS, the cells were incubated with secondary antibody labeled with Cy3 (1:200; cat. no. A0516, Beyotime) at room temperature in the dark for 60 min, and counterstained with DAPI (Aladdin). Finally, the glass slide were mounted with anti-fading reagent (Solarbio, Beijing, China), and the cells were observed under a fluorescent microscope (BX53, Olympus) at 400× magnification.

Following quick fixation in 4% paraformaldehyde solution for 24 h, the samples were subjected to gradient dehydration (from 50% to 100% ethanol). After that, the samples were cleared with xylene, fixed in paraffin, and cut into 4 µm thick sections to mount onto slides. After deparaffinization and rehydration, the slides were warmed to 60 °C for 1 h in a dry oven to soften the paraffin. For ten minutes each, the slides were washed in xylene I, xylene II, 100% ethanol, 95% ethanol, 90% ethanol, and 80% ethanol. After washing the slides with deionized water for 5 min, they were heated in an oven at 95–100 °C for 20 min in an antigen retrieval solution. The sections were removed from the oven after antigen retrieval and allowed to cool at room temperature. After three washes with PBS, the tissue the sections were covered with goat serum and incubated at room temperature for 1 h. After blocking, the samples were incubated with the indicated antibodies overnight at 4 °C, washed with PBS three times, and incubated with corresponding secondary antibodies for 1 h at room temperature. After washing with PBS three times, the samples were counterstained with DAPI (Beyotime, Shanghai, P0131) and mounted with anti-fading reagent (Solarbio, China). Representative images were photographed using a confocal microscope (Nikon A1R, Tokyo, Japan).

The mouse primary trophoblast cells were identified by immunofluorescent staining of cytokeratin 7 (CK7). The cells were cultured on glass slides, and fixed with 4% (m/v) paraformaldehyde (Sinopharm, Shanghai, China) for 15 min. After rinsing with PBS, the cells were permeated with 0.1% (m/v) TritonX-100 (Beyotime, Shanghai, China) for 30 min, rinsed with PBS, blocked with 1% bovine serum albumin (Sangon, Shanghai, China) for 15 min, and incubated with antibody against CK7 (1:50, Cat no., A4357, ABclonal, Wuhan, China) at 4 °C overnight. Subsequently, the cells were incubated with goat anti-rabbit IgG labeled with Cy3 (1:200, Cat no., A27039, Invitrogen, Carlsbad, CA, USA) at room temperature for 60 min in the dark, followed with counterstaining of DAPI. Finally, the glass slides were sealed with anti-fading reagent (Solarbio, Beijing, China), and images were taken with a fluorescent microsope at magnification 400 ×.