Harris haematoxylin solution

Cells on membrane inserts were fixed in 4% (w/v) formaldehyde before transfer to 70% EtOH. Staining was carried out according to the manufacturer's instructions [Periodic Acid-Schiff (PAS) staining system, Sigma]. Cells on membranes were first immersed in the periodic acid Solution for 5 min before rinsing in three changes of distilled water. The membranes were then placed in Schiff's reagent for 15 min before rinsing in running tap water for 5 min. The membranes were counterstained in Harris Haematoxylin solution (Sigma) for 60 s. Counterstained membranes were rinsed in running tap water then dried by blotting on tissue and mounted using Hydromount™ mounting solution (National Diagnostics). Slides were dried overnight before imaging with an Olympus IX71 inverted fluorescence microscope equipped with a 40× objective and cellSens software (Olympus).

The formalin-fixed tissue sections were dewaxed in Citroclear (HD supplies, Aylesbury, UK) for 15 min and rehydrated through absolute alcohol to 70% alcohol (1 min in each concentration of alcohol). The sections were briefly washed in distilled water and stained in Harris Haematoxylin solution (Sigma-Aldrich) for 4 min. After staining, the sections were washed in running tap water for 1 min followed by differentiation in 1% acid alcohol for 10 s. The sections went through washing and bluing in running tap water for 5 min before eosin staining. After staining using eosin solution (Sigma-Aldrich) for 30 s, the sections were washed in tap water for 1 min. Dehydration was performed whereby the sections were taken through changes of 70% alcohol through to absolute alcohol. Before mounting the sections were immersed in Citroclear (HD supplies) for 30 s. The sections were finally mounted with DEPEX mounting media (Thermo Fischer Scientific, Hemel, UK) and sealed. Slides were viewed using a Nikon Eclipse E400 microscope (Nikon, Japan) equipped with lenses at × 20 and × 40 magnification. The average of villus heights was determined by measuring the length of 10 to 15 villi from three different fields of view per specimen using NIH Image software (ImageJ, version 1.45 s, https://imagej.nih.gov/ij/docs/index.html). All slides were analysed in a blinded fashion.

Embryos were fixed overnight in Bouin’s solution (Sigma), dehydrated in an ethanol series and embedded in paraffin-wax. Seven micron sections were stained using Harris’ haematoxylin solution and 2% Eosin Y (both Sigma). Images were captured on an Axiophot2 upright microscope.