Gill no 3

After 7 days of culture, 3D gels were rinsed with PBS and fixed in 10% formaldehyde for 20 minutes. Gels were then detached from the bottom of wells and washed in distilled water. After 5 minutes of incubation in 60% isopropanol, gels were stained for 2 minutes with freshly filtered Oil-Red-O (ORO) [working solution made of 3:2 vol/vol dilution of ORO stock (3 mg/ml in 99% isopropanol) and distilled water]. The gels were then rinsed in water and counterstained with hematoxylin (Gill No. 3, Sigma-Aldrich, UK) for 30 seconds, followed by several long washes in tap water. Gels were topped with a 50% glycerol in Tris-buffered saline (TBS) solution before observing and imaging. The ORO staining was quantified by counting ORO positive cells directly under the microscope (Leica DMIL, 20x objective). A cell was deemed “ORO positive” when harbouring 10 or more lipid droplets in its cytoplasm. A minimum of 100 cells were evaluated from 4~5 fields in each gel.

For immunohistochemistry, the uterine were stored in 4% paraformaldehyde and embedded in paraffin. Serial slides with about 6 μm were prepared, deparaffinized in xylene, rehydrated with different concentrations ethanol, and rinsed with water. Endogenous peroxidase was blocked with 3% hydrogen peroxidase. Slides were treated with chondroitin ABC lyase (0.15 U/mL) and blocked with 10% normal goat serum for 1 hour. Expressions of cytokeratin, vimentin, integrin α_νβ₃, and leukemia inhibitory factor (LIF) proteins were performed by incubating slides of rat uteri with rabbit polyclonal antibodies against cytokeratin, vimentin, integrin α_νβ₃, and LIF overnight at 4°C. Slides were incubated with secondary antibodies at 1 : 3000 dilution followed by DAB solution for 1 hour. Then, slides were briefly stained with hematoxylin solution (15 seconds) (Gill no. 3; Sigma) and evaluated by a microscope (Nikon).

Tissue fixation, dehydration, paraffin embedding, and hematoxylin and eosin (H&E) staining were performed as previously described^{44 (link)}. Briefly, harvested tissues were fixed in 10% formalin, dehydrated, paraffin embedded. Five-micron sections were stained with the custom αhPRLrI primary antibody (New England Peptide) at a 1:12,500 titer, following an additional 10% formalin fixation blocking step prior to antigen retrieval. After washing, slides were incubated with SignalStain Boost IHC Detection Reagent (8114 S, Cell Signaling) for 1 h, followed by incubation with SignalStain DAB Substrate (8059 S, Cell Signaling) for 1 min. Slides were counterstained with hematoxylin (Gill No. 3; GHS332, Sigma), dehydrated, and coverslipped using Permount mounting medium (50–277–98, Electron Microscopy Sciences). hPRLrI staining was expressed as an Allred score, namely the sum of the proportion of positive cells (0–5 scale) and mean intensity (0–3 scale)^{44 (link)}.