Troma 1

Implantation sites were fixed in 10% formalin overnight, embedded in paraffin, 8 μm sections were rehydrated, antigen retrieval was performed, non‐specific binding was blocked, and specific immunostaining was conducted as described below. In the case of Crry staining, antigen retrieval was performed in 10 mmol/L citric acid (anhydrous; Sigma‐Aldrich), 0.05% Tween‐10, pH 6.0 in a pressure cooker for 3 minutes. Blocking and staining were performed in 1% BSA, 10% donkey serum, and 5% mouse serum in PBS. Rabbit anti‐Crry (1:1000; provided by V. Michael Holers, Division of Rheumatology, University of Colorado School of Medicine, Aurora, CO) was placed in blocking buffer overnight. Donkey anti‐rabbit HRP (Jackson Immunoresearch) was used at 1:200. Staining was visualized with diaminobenzidine (DAB).
For trophoblast staining, 7.5 dpc implantation sites were collected as described above. Antigen retrieval was accomplished with 10 mmol/L Tris‐EDTA pH 9.0 for 3 minutes in a pressure cooker. Staining was accomplished with TROMA‐I (1:50 dilution of hybridoma supernatant; Developmental Studies Hybridoma Bank, University of Iowa) and goat anti‐rat light chain horseradish peroxidase (HRP). Staining was visualized with DAB. Controls employed a second Ab only.

Tissues were fixed in formalin or EAF (ethanol, acetic acid, and formalin mixture) and paraffin‐embedded, followed by hematoxylin and eosin (H&E) staining according to routine protocols. For the postmortem analysis of lung metastases, paraffin‐embedded lungs were sectioned and H&E‐stained at five different levels throughout the lungs. Immunohistochemistry was performed using rabbit anti‐cytokeratin‐5 (1:500, PRB‐160P; Covance, Princeton, NJ, USA), rat anti‐cytokeratin‐8 [1:1500, Troma‐I; Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA, USA], rabbit anti‐ERα (1:1000, sc‐542; Santa Cruz Biotechnology, Dallas, TX, USA), and rabbit anti‐PR (1:300, RM‐9102; Thermo Fisher Scientific, Waltham, MA, USA).

Colonic frozen sections (4 μm thick) were stained with anti-Ki67 (Abcam, Cambridge, MA, USA), rabbit anti-CHI3L1 (Affinity Bioreagent, Golden, CO), rat anti-F4/80 (Serotec, Raleigh, NC, USA), -CD4 (eBioscience, San Diego, CA, USA), -CD11b (BD Pharmingen, San Jose, CA, USA), or -cytokeratin 8, TROMA–1 (Developmental Studies Hybridoma Bank at the University of Iowa (Iowa City, IA) antibodies, as well as normal rabbit Ig using the avidin-biotin-complex system (Vector laboratories) as previously described [12 (link)].