Immunoelectron Microscopy of Human Placenta

Immunoelectron microscopy (immuno-EM) was performed at the Biocenter Oulu Electron Microscopy Core Facility as described previously.^{37 (link)} In short, fresh human placental samples from SPTBs and STBs were fixed and cut with a Leica EM UC7 cryoultramicrotome (Leica Microsystems, Vienna, Austria). For immunolabeling, sections of Butvar-coated nickel grids were exposed to primary antibody to HSPA5 (3177, 1:100 dilution; Cell Signaling Technology) and bound antibodies were labeled by incubation with protein A–conjugated 10 nm gold (Cell Microscopy Core, University Medical Center Utrecht, The Netherlands). Controls were prepared by replacing the primary antibody with PBS. To reduce background labeling, endogenous immunoglobulins were blocked using Fab fragments (Goat Anti-Human IgG [H + L]; Jackson ImmunoResearch Europe Ltd, United Kingdom). Samples were incubated with Fab fragments for 30 min after primary blocking step before incubation with primary antibody. Thin sections were examined with a Tecnai G2 Spirit 120 kV transmission electron microscope (FEI, Eindhoven, The Netherlands), and images were captured by a Quemesa CCD camera (Olympus Soft Imaging Solutions GMBH, Münster, Germany).