E15.5 Mouse Embryo Micro-CT Using a Bruker Skyscan 1172 Micro-CT

Materials and Reagents

1. Mouse mating and embryo dissection

   1. Petri dishes (100 mm) (BD Biosciences, catalog number: 351029 or similar)

   2. 35 mm cell culture dishes (BD Biosciences, catalog number: 352096 or similar)

   3. Sterile disposable tubes (50 mL) (BD Biosciences, catalog number: 352070 or similar)

   4. 15 mL tubes

   5. Mice

   6. Pure ethanol (Sigma, catalog number: 493546)

   7. Phosphate-buffered saline (PBS), without Ca²⁺ and Mg²⁺ (Life Technologies, Gibco^®, catalog number: 10010)

   8. 4% paraformaldehyde (PFA) (Santa Cruz Biotechnology, catalog number: sc-281692)

   9. 70% ethanol (see Recipes)




2. Micro-CT sample preparation

   1. Lugol’s solution (Sigma, catalog number: 32922)

   2. Tween-20 (Sigma, catalog number: P1379)

   3. Staining solution with contrast agent (see Recipes)

Equipment

1. Blunt forceps (FST Standard Pattern forceps or similar)

2. Fine forceps (Dumont #5 or similar) and scissors (FST, catalog number: 14060-09 or similar)

3. X-Ray computed microtomography Skyscan 1172 (Bruker)

   This instrument is a desktop µCT equipped with a fully distortion-corrected 11 MP X-ray camera (a 12-bit cooled CCD camera coupled to scintillator). The X-ray source is 20–100 kV, 10 W, with a <5 µm spot size. 0.8 µm is the highest resolution (only for small samples) and 25 µm is the lower resolution. The maximum object size is 50 mm. Skyscan 1172 is equipped with three filter positions (None, 0.5 mm Al, or Al+Cu).

Software

1. DataViewer 1.5.4.6 (Bruker)

2. NRecon 1.4.4 (Bruker)

3. CTVox 3.3.0 (Bruker)

Procedure

1. Mice mating

   1. Keep mice on a 12:12 h light/dark cycle with unrestricted access to food and water. Use animals aged 2–4 months for mating.

   2. For mating, put a male and two females in a cage after 6 pm.

   3. Before 9 am on the next day, check for the formation of vaginal plug: lift a female by her tail, slightly dilate the vaginal opening with a blunt forceps, and check for a white mass.

   4. Consider the day of plug detection as day E0.5. House the mated females separately from the male. Repeat mating procedure with non-mated females the following days.




2. Embryo dissection

   Embryo dissection is performed as described in Zeeb et al. (2012).

   1. At day E15.5, euthanize the pregnant female.

   2. Place the mouse on a supine position and spray 70% ethanol on the abdomen.

   3. Make a V-formed incision on the skin and abdominal cavity and lift the tissue to expose the internal organs.

   4. Identify the V-shaped uterus with the string of embryos inside, cut at the proximal (cervical) and distal (ovaries) ends, and remove the uterus to a tube with ice-cold PBS.

   5. Dissect the embryos in a dish with PBS on ice. First, cut the uterus into single embryos-containing pieces. Place each embryo in a 35 mm dish with PBS and remove the uterine tissues and yolk sac. Keep yolk sac membranes for genotyping if required.




3. Embryo fixation

   1. Place single embryos in 15 mL tubes with 5 mL of 4% PFA and incubate at 4 °C for 24 h.

   2. After fixation, keep the embryos in PBS at 4 °C. If needed, the samples can be kept at 4 °C for several days.




4. Micro-CT sample preparation

   1. Prepare fresh staining solution of 20% Lugol’s solution + 0.5% Tween-20 pH 7.3.

   2. Keep samples in staining solution in constant gentle agitation.

   3. Change solution daily for 10 days.




5. Micro-CT acquisition

   Perform acquisition at 80 kV using a 0.5 mm Al filter at a resolution of 3 µm, 0.4° of rotation step, 360° scan, and 4× frame averaging.




6. Micro-CT image reconstruction

   Obtain image reconstruction using NRecon software (Bruker), with proper misalignment compensation, no smoothing, 5 ring artifacts reduction, and 30% beam-hardening correction.

Data analysis

1. Open dataset of the single embryo in DataViewer software (Figure 1).

   
   

   
   Figure 1. A screenshot of DataViewer software. An embryo is visualized in virtual sections in three planes.

   
   

2. Select the plane by moving along the x- and y-axes. In our case, we were interested in performing the morphometric analysis of the embryo heart, in particular to measure the width of the ventricular walls and interventricular septum and the area of the pericardial cavity. The same plane, the best at representing the features we were interested in, was selected in all analyzed embryos.

3. Measure the features of interest by right-clicking to obtain the length in μm.

4. To perform a qualitative investigation, the dataset could be visualized in 3D by using CTVox software (Figure 2 and Video 1).

   
   

   
   Figure 2. A screenshot of CTVox software. A E15.5 embryo is visualized in 3D slicing.

   
   
   
   Video 1. Embryo

Recipes

1. 70% ethanol

   Obtained by mixing 70 mL of pure ethanol with 30 mL of water.




2. Staining solution with contrast agent

   Staining solution with contrast agent is obtained by making a 20% Lugol’s solution with 0.5% Tween-20 and adjusting pH to 7.3.

Acknowledgments

This protocol was adapted from the previously published study (Astanina et al., 2022). This work was supported by AIRC – Associazione Italiana Per la Ricerca sul Cancro (grants 22910), Regione Piemonte (grant A1907A, Deflect), Fondazione CRT, Ministero dell’Università e della Ricerca (PRIN 2017, grant 2017237P5X), FPRC 5xmille 2016 MIUR (Biofilm), and ERA-Net Transcan-2 (grant TRS-2018-00000689) to F.B. and Ministero dell’Università.

Competing interests

The authors declare no conflict of interests.

Ethics

All animal procedures were approved by the ethics committee of the University of Turin and by the Italian Ministry of Health (protocol approval no. 864/2015‐PR).

References

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