Combined IBA1 IHC and XY FISH staining

Selected cases were costained with IBA1 IHC and XY FISH to characterize the donor cells. Sections were first incubated with anti-IBA1 antibody (Wako Chemicals, Japan), then with poly horseradish peroxidase (HRP)-conjugated goat anti–rabbit immunoglobulin G secondary antibody (Invitrogen, Carlsbad, CA), and visualized using Alexa Fluor 488 tyramide reagent (Invitrogen) per manufacturer instructions. The tyramide reagent forms a stable, covalently ligated fluorochrome. The slides were imaged using a TissueFAXS. The same sections were then stained for XY FISH (as previously described) and reimaged using a TissueFAXS. Because some of the IHC signal was lost following FISH, digital scans of IBA1 IHC were overlaid onto FISH images using DAPI-stained nuclei for orientation (Adobe Photoshop).