Transmission electron microscopy
This protocol is extracted from research article:
Reversible EMT and MET mediate amnion remodeling during pregnancy and labor
Sci Signal, Feb 11, 2020; DOI: 10.1126/scisignal.aay1486

Fetal membranes from cesarean and vaginal deliveries and fetal membrane explants from normal term pregnancies with or without CSE exposure were fixed, stained, and embedded in Poly/Bed 812. Initial fixation was for 24 hours at 4°C in a fixative with 2.5% paraformaldehyde, 0.2% glutaraldehyde, and 0.03% picric acid in 0.05 M cacodylate buffer. After fixation, samples were rinsed three times with cacodylate buffer and postfixed with 1% osmium tetroxide in 0.1 M cacodylate buffer. Sonicated tissue was rinsed twice with deionized water and stained en bloc with 2% aqueous uranyl acetate for 1 hour at 60°C. The samples were then dehydrated by a series of ethanol-water solutions (50, 75, 95, and 100% ethanol for three exchanges). Dehydrated tissue was infiltrated with two exchanges of propylene oxide, then with propylene oxide–diluted Poly/Bed resin at 1:1 ratio and 1:2 ratio, and then twice with pure Poly/Bed 812. Last, the samples were embedded in Poly/Bed 812 and cured overnight at 60°C. Because precise tissue orientation could not be maintained during curing of the resin, the first resin blocks were cut to give a wide flat face for the desired sectioning plane, replaced into new embedding molds, and cured again. Samples were cut as 90-nm sections, placed on Formvar-coated slotted grids, and poststained for 3 min with a solution of Reynold’s lead citrate. Images were taken with a JEM-1400 electron microscope (magnification, ×4000 or ×1500; JEOL) (63). This value was used to calibrate the tight junction length from pixels to nanometers.

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