Nucblue fixed readyprobes reagent

Immunocytochemical staining for CD45 (Abcam; ab30470; 1:200), Vimentin (Abcam; ab92547; 1:500), Cytokeratin (CK)-18 (Abcam; ab668; 1:800), HLA-G (Abcam; ab52455; 1:200), and HLA-DR (Abcam; ab929511; 1:50) were used to monitor cell types and document their immune regulatory status. Antibodies were titrated to determine appropriate dilutions to ensure specific and uniform staining. After 24 hours, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton-X, and blocked with 3% bovine serum albumin in 1× PBS, before incubation with primary antibodies overnight. Cells were washed three times in 1× PBS and then incubated with species-specific secondary antibodies (1:1000) for 1 hour. The MPSs were washed with 1× PBS, and then treated with NucBlue^® Fixed ReadyProbes Reagent (R37606; ThermoFisher Scientific, Waltham, MA) to stain the nucleus. Primary and secondary concentrations were validated based on previous chip-based studies.

Each chamber of the OOC devices were staining with individual cell-specific antibodies in order to document that cells on-chip maintained in utero characteristics. Immunocytochemical staining for cytokeratin (CK)-7 (ab9021; Abcam, Cambridge, MA, USA), E-cadherin (ab15148 Abcam, Cambridge, MA, USA), and MUC18 (ab75769; Abcam, Cambridge, MA, USA) was performed after 24 h, as previously described.^{46 (link),47 (link)} Manufacturers’ instructions were followed for determining appropriate dilutions of antibodies to ensure specific and uniform staining. After 24 h, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X, and blocked with 3% bovine serum albumin in 1× PBS, before incubation with primary antibodies overnight at 4°C. After washing with 1× PBS, the PLA-OOCs were incubated with Alexa Fluor 488–, 594–, and 647–conjugated secondary antibodies (Thermo Fisher Scientific) diluted 1:400 in 1× PBS for 2 h in the dark. The PLA-OOCs were washed with 1× PBS, and then also treated with NucBlue^® Fixed ReadyProbes Reagent (R37606; Thermo Fisher Scientific, Waltham, MA) to stain the nucleus. OOCs were fixed in 4% paraformaldehyde for 15 min and stained using the Masson Trichrome method to identify collagen within the microchannels. Three microscopic fields for each condition were captured at 10x.