Antifade solution

Spheres and organoids were fixed in 4% of paraformaldehyde for 1 h at room temperature. Alcohol was used for a dehydrated purpose and followed by acetone and isopropanol for 20 min at room temperature. The dehydrated spheres and organoids were embedded in the paraffin block and sectioned with a 5-μm thickness. Immunofluorescence staining was carried out with rehydrated sections and treated with antigen retrieval solution (Dako). Then we blocked the sections with 1% BSA and 2% FCS in PBS for 1 h. Then the sections were incubated with primary antibodies diluted 1:100 for 1.5 h. Then we washed the sections with PBS with 0.1% Tween 20 and incubated with secondary antibodies for 1 h. Finally, the sections were washed with PBS with 0.1% Tween 20 and mounted in Antifade solution (Sigma-Aldrich).

Tissue sections were deparaffinized and rehydrated through xylene, gradient ethanol, and distilled water. After rinsing three times in PBS, the tissue sections were incubated with 1 μmol/L Hoechst-33258 (Sigma) for ten minutes. After washing three times in PBS, slides were transferred to coverslip with antifade solution (Sigma), and then analyzed by fluorescence microscopy. Cells with condensed chromatin or fragmented nuclei emitting intense blue fluorescence were classified as apoptotic.

Collected embryos (1 dpc) of both groups were cultured up to the morula stage as indicated in section Embryo Culture and TNFα Treatment. Randomly selected morulae from both groups of embryos (n = 10) were stained with annexin V and propidium iodide (Annexin-V-FLUOS Staining Kit, Roche Diagnostics, Poland) to confirm the expression of phosphatidylserine as an early sign of apoptosis induction in blastomeres in embryos (28 (link), 30 (link)). Briefly, the embryos were transferred to freshly prepared and preheated microdrops of PBS supplemented with Ca²⁺ Mg²⁺, supplemented with bovine serum albumin (BSA; Sigma, Poland). The embryos were washed three times, each time in a fresh microdrop of PBS (100 μL). Afterward, they were stained at room temperature for 10 min with the Annexin-V-FLUOS (Sigma, Poland) solution reagent with the addition of propidium iodide (20 μL of working-strength solution). After that, the embryos were washed in 10 μL microdrops of fresh PBS with Ca²⁺ Mg²⁺. Before the evaluation under a fluorescence microscope (Leica), the embryos were transferred into microdrops of fresh PBS with the addition of an anti-fade solution (Sigma, Poland). The observations were performed immediately after completed staining.