Fish skin gelatine

First trimester placental and decidual tissues (6^th - 12^th week of gestation) were fixed in 7.5% (wt/vol) formaldehyde and embedded in paraffin. Serial sections (3 or 30 μm) were deparaffinised in Xylol (10 min) and rehydrated in a decreasing series of ethanol (100%, 90%, 70%, 0%; 1 min each step). Antigen retrieval was performed using 1× PT module buffer 1 (pH 6, Thermo Fisher Scientific) for 35 min at 93°C using a KOS microwave histostation (Milestone). Sections were blocked using 0.05% cold water fish skin gelatine (Sigma-Aldrich) for 30 min at room temperature and incubated overnight at 4°C with primary antibodies in PBS with 0.05% fish skin gelatine (see antibody list). Secondary antibodies were incubated for 45 min at room temperature in PBS with 0.05% fish skin gelatine and 1 μg/ml 4′,6-Diamidin-2-phenylindol (DAPI, Roche). Finally, tissue sections were mounted with Fluoromount-G (Thermo Fisher Scientific) and covered. Images were acquired with a fluorescence microscope (Olympus BX50 equipped with Cell^P software) or with a confocal laser scanning microscope (LEICA, TCS SP8X, equipped with Leica LAS AF software). Confocal images are depicted as maximum projection of total z-stacks and brightness and contrast were adjusted in a homogenous manner using Leica LAS AF.

Total cell extracts equivalent to 1 × 10⁶ cells were separated on SDS-polyacrylamide gels and subject to either standard or LICOR western blotting analysis according to the manufacturers’ instructions. For standard western blots, duplicate gels were generated and one was stained with Coomassie and the other was used to produce the nitrocellulose blot. Blots were blocked in 5% milk in TBST and washes were performed in TBST (0.05% Tween). Blots were then probed with 1/1000 α-gLUC primary antibody (New England Biolabs) and 1/2000 α-rabbit secondary antibody (Bio-Rad). For LICOR blots, blocking was performed in 50 mM Tris, pH 7.4, 0.15 M NaCl, 0.25% BSA, 0.05% Tween, 2% fish skin gelatine (Sigma, UK) and washes were performed in TBST (α-gLUC) or PBST (α-GFP). Nitrocellulose blots were incubated with 1/1000 α-gLUC or 1/5000 α-GFP (Life Technologies) and 1/20 α-tubulin (kind gift from Keith Gull), followed by 1/10,000 α-mouse and 1/10,000 α-rabbit IR Dye antibodies (LICOR). Lysates for gLUC assays were prepared by adding 20 µl of 1 × luciferase cell lysis buffer (New England Biolabs) to 2 × 10⁶ pelleted cells and these were directly used to perform BioLux Gaussia luciferase assays (New England Biolabs) according to the manufacturers’ instructions and using a TopCount plate-reader with white-walled plates. One-way ANOVA tests were carried out in GraphPad Prism (version 7).