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Determination of the windows of lethality

Purpose

To evaluate embryonic viability, it is first important to determine the developmental window of lethality as it can provide valuable information about the type of defects present in the embryos and/or placentas. To determine the window of lethality, representative stages will be chosen to dissect the embryos or fetuses. These stages represent critical time points in development and the results will help inform subsequent histological analyses. The embryos are sampled for macroscopical analyse and viability at times throughout pregnancy with focus on development of particular organs between E9.5 and E18.5.

To dissect embryos/fetuses at precise stages, parents intercrosses are followed rigorously to check the vaginal plugs (formed by the polymerization of proteins derived from prostatic secretions) each morning. The morning at which a vaginal plug is noted corresponds to the stage 0.5 dpc (days post-coïtum) or E0.5 (Embryonic day 0.5). Embryos and fetuses will be genotyped by using extraembryonic membranes (yolk sac) or tail pieces, respectively.

Equipment

  • Stereomicroscopes 
  • Automated macroscope equipped with digital camera
  • Hot plate for viability test of E18.5 fetuses.

Recommendations

For statistical reasons, at least 28 embryos have to be dissected and genotyped for one developmental stage in order to know if the line is lethal, viable or subviable. This corresponds to the dissection of four pregnant females (7 pups/female approximately).

Characterization of embryonic and perinatal defects

Purpose

To detect developmental defects in prenatal mice from E5.5 to E18.5. Embryos and fetuses are analyzed macroscopically, then fixed in Bouin's fluid, decalcified (for ages E16.5 and beyond), embedded in paraffin, serially sectioned at 7 µm and stained with hematoxylin and eosin or Mallory's trichrome and hematoxylin stain. Whole skeletal analyses can be performed on E18.5 fetuses (alcian blue and alizarin red staining)

Equipment

Stereomicroscopes

Slide scanner (Nanozoomer 2.0 HT, Hamamatsu Photonics K.K., Hamamatsu City, Japan)

Microtomes

Cryostats

Recommendations

Always use age-matched littermates as controls

Characterization of gene expression pattern

LacZ enzyme activity is evaluated by histochemical staining in order to characterize anatomical structures expressing the targeted gene in embryos carrying a lacZ transgene. See gene expression pattern.

Micron-scale Computed Tomography (µCT) of Embryos

Purpose

Embryos may be morphologically examined in situ using micron-scale X-ray Computed Tomography (also called µCT). This can be an effective strategy for screening a large number of different groups (e.g. genetic screens, multiple alleles, or drug treatments) for organ-specific or broad effects on embryo anatomy, relieving extensive embryo processing of standard histological techniques.

Equipment

µCT (PerkinElmer Quantum FX, Waltham , Massachusetts)

Sample Results

Embryo analysis by HREM

Purpose

HREM is an effective and powerful tool for assessing structural abnormalities in embryos. Three dimensional reconstruction of embryo slices provides the definition of histological staining with the completeness of 3-D imaging.

Equipment

HREM imaging is operated with a home made system (developed with the IGBMC Imaging Center)

Anapath_EmbryoAnalysis_HREM equipment.png

 

Sample processing

1-The sample is fixed in Bouin’s solution  

2-JB4 Resin embedding :

  • Ethanol dehydration
  • Resin infiltration
  • Resin polymerisation
  • Bloc drying
  • Bloc storage

3-Samples limitations: the size must not exceed 1.5 cm3

Sample results

Imaging_HREM Embryos.png

Imaging-HREM-Embryos Organs.png

Recommendation

It is recommended to use at least 3 experimental and 3 control samples for analyse.

Developmental markers


Purpose

To better characterize primary defects of mutant embryos or to screen for abnormalities, vascular (PECAM), neural (neurofilament) markers are proposed. Other marker stainings can be developed on demand.

Deliverables

  • Macroscopic and microscopic images of the abnormalities
  • Fixed embryos or fetuses
  • Embryos, fetuses or specific embryonic tissues fixed in liquid nitrogen for RNA analysis
  • Sets of stained slides
  • Pathology reports with expert opinion 
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