Pannoramic 250 microscope slide scanner

Following harvest, placental tissue samples (GD15, 16 and 21) were fixed in 3 mL 10% neutral buffered formalin and then embedded in paraffin wax. Placental sections (5 μm; Leica RM2245 Semi-automated Rotary Microtome; Leica Biosystems, U.K.) were stained with haematoxylin and eosin (Sigma-Aldrich, U.K.). Whole sections (3 per slide) were imaged using brightfield microscopy (3D-Histech Pannoramic-250 microscope slide-scanner) at the Bioimaging Facility, University of Manchester. Quantification of placental area, comprising the junctional and labyrinth zones, was performed using ImageJ (NIH, U.S.A.) at 1× magnification per section.

Colons were excised from animals and prepared for histology.³⁴ In brief, colons were flushed and fixed with modified Bouin's solution (50% ethanol, 5% acetic acid in distilled water) using a syringe and gavage needle. The intestinal section was then opened longitudinally, rinsed briefly in PBS and rolled from the proximal end into a “swiss roll”, before transferring to a tissue‐processing cassette. The sample was placed in 10% buffered formalin overnight (RT) and then dehydrated through ethanol and xylene before being embedded in paraffin blocks and sectioned at 5 μm thickness (Leica RM2255 microtome). Sections were mounted onto X‐tra™ Adhesive slides (Leica). Hematoxylin and eosin staining was performed using standard protocols. Images were taken using the 3D‐Histech Pannoramic‐250 microscope slide‐scanner. Histology images were processed using the opensource software FIJI.³⁵