PROTOCOLS

Placental analysis

Placentas from homozygous mutant lines are analysed at E9.5 and E14.5 by histological staining.

Sample preparation

Placentas are dissected at E9.5 and E14.5, fixed in 4% paraformaldehyde and transferred into 1xPBS. If not immediately embedded, the samples are then stored at 4C.

E14.5 placentas are manually cut in half along the sagittal midline of the placenta, identified by the insertion of the umbilical cord. E9.5 placentas are left intact.

The tissues are processed for paraffin histology by sequential incubations in:

  • 30%, 50%, 70%, 95% ethanol (in motion, 30 minutes at RT for each concentration)
  • 100% ethanol twice (in motion, 1 hour at RT, and overnight at 4C)
  • 100% Xylene twice (in motion for 1 hour at RT)
  • melted paraffin three times (at 60C for at least 30 minutes)

The tissues are then embedded in paraffin blocks.

In each block, 3 wild-type and 3 mutant placentas are placed side-by-side, after having been processed in parallel. Serial 7 µm sections are cut using a Leica paraffin microtome, mounted onto Polysine slides and left to dry.

Staining

For E9.5 embryos every 6th section is chosen for haematoxylin and eosin (H&E) staining, which is performed according to standard protocols. For E14.5 embryos every 20th section is chosen. The intermediate sections are retained to allow repeat results if necessary.

Sections are de-paraffinised by immersing in:

  • 100% Xylene for 10 minutes at RT
  • 100% ethanol twice (10 minutes each at RT)
  • 70%, 50% and 30% ethanol (for 5 minutes at RT for each concentration)
  • 1xPBS wash

Sections are incubated in Mayer’s Haematoxylin (Sigma, #51275) for 10 minutes and briefly washed in tap water. They are then differentiated in 70% acid alcohol (70% ethanol, 1% hydrochloric acid) for 10 seconds, followed by incubation in cold tap water for 10 minutes.

Sections are incubated in Eosin (alcoholic Eosin, Sigma #HT110116) for 30 seconds, followed by an increasing ethanol row (70% for 1 minute, 100% for 1 minute, 100% for 1 minute). The sections are then incubated in 100% xylene for 2 minutes before mounting with Eukitt quick-hardening mounting medium (Sigma #03989).

Analysis

H&E stained sections through the midline of the placenta are scanned using a Hamamatsu slide scanner and scored for phenotypes.

Placental analysis is performed by the Hemberger Lab at the Babraham Institute.