Tissue contrasting for µ-CT scanning

Tissue contrasting for µCT scanning using PTA (Phosphotungstic

Acid)

1.       Optional step: before dissecting the embryos, do perfusion of the mother first with 40ml PBS to remove the blood and then perfuse with 40ml of freshly mixed 4%PFA (paraformaldehyde) in PBS (pH=7.4). This step is not necessary, nevertheless, we have noticed slightly better results.

2.       Dissect the embryos, wash them for 1min in an ice cold PBS (on a 10cm dish or in a falcon tube, make sure the samples do not get dry during the manipulations) and place them into the 15ml or 50ml falcon tube with freshly prepared 4%PFA in PBS (pH=7.4). The tube has to be filled with PFA till the very top. In general, the volume of PFA must be at least 10x the volume of the embryo/sample. That means that you can use 15ml falcon tube for incubation of small (eg E12.5) embryos but use 50ml falcon tube for incubating bigger size (eg E15.5) embryos.

3.       Fix the embryos in 4%PFA overnight, with slow rotation, at +4˚C.

4.       Discard the PFA and replace it with 30% ethanol (this is the start of the dehydration), incubate for 24 hours, at +4˚C, slow rotation.

5.       Replace the 30% ethanol with 50% ethanol and repeat the 24 incubation (with the same conditions as in step 4)

6.       Replace the 50% ethanol with 70% ethanol and repeat the 24 incubation (with the same conditions as in step 4)

7.       Replace the 70% ethanol with 90% ethanol and repeat the 24 incubation (with the same conditions as in step 4)

8.       Remove the 90% ethanol and place the embryo in the mixture of 1.0-1.5% PTA in 90% methanol, incubate for several days (details below) with slow rotation, at +4˚C; seal the tube well (for instance with parafilm) to avoid leaking; tube has to be again filled till the very top.

Details for incubation times: E12.5 embryos were stained for 7 days in 1.0% PTA, E15.5 embryos for 3 weeks in 1.5% PTA and E18.5 embryos for 7 weeks in 1.5% PTA. Important note: starting with the embryonic stage E15.5 onwards, it is necessary to allow the solution to get a better access to the inner tissues, therefore we separated the body from the head at the level of shoulders and incubated only the head. In the case that the whole body is needed for scanning, we suggest perforating the skin and skull with needles at several locations to allow the PTA mixture to get inside the sample but this has not been tested by us.

9.       When the contrasting time is over, replace the solution (1.0-1.5% PTA in 90% methanol) with 90% ethanol and repeat the 24 incubation (with the same conditions as in step 4; this is the start of rehydrating the sample)

10.   Replace the 90% ethanol with 70% ethanol and repeat the 24 incubation (with the same conditions as in step 4)

11.   Replace the 70% ethanol with 50% ethanol and repeat the 24 incubation (with the same conditions as in step 4)

12.   Replace the 50% ethanol with 30% ethanol and repeat the 24 incubation (with the same conditions as in step 4). After this the rehydration of the sample is complete.

Additional notes:

For the purpose of scanning using microCT scanner where the sample is rotating inside the scanner, to avoid movement of the sample, we embedded the sample in 0.5% agarose gel (A5304, Sigma-Aldrich) in polypropylene conical tubes (0.5, 1.5 or 15 ml depending on the sample size to minimize the amount of medium surrounding it.

For a CT instrument where sample is not moving during the scanning, we recommend the same fixation to avoid micro-movement due to the tissue drying and shrinking.

If the sample is not stained sufficiently after the suggested time (note that the times of tissue contrasting for various embryonic stages are designed for contrasting the head region sufficiently, we have not made any analysis on the body), it is possible to repeat/extend the procedure starting with the step 4).

We have noticed that using freshly mixed solutions (both PFA and PTA) improves the quality of the contrasting procedure. It is acceptable to prepare 4%PFA in PBS (ph=7.4) and store it in 50ml falcon tubes at -20˚C for the use during the next two months. PTA has to be always prepared fresh. For longer staining times (over 1 week), we recommend to change the PTA solution weekly for a fresh mixture.

Be aware of the health risks associated with the use of this chemical (!), we recommend the use of chemical fume hood during all manipulations and, obviously, disposable nitrile gloves and a lab coat. Be aware of special regulations when discarding the PTA contrasting mixture.

For more information on the scanning and segmentation procedure, please, see our publication: https://iopscience.iop.org/article/10.1088/1748-0221/11/03/C03006/pdf

“Use of micro computed-tomography and 3D printing for reverse engineering of mouse embryo nasal capsule” by Tesarova et al, 2016, JINST.

For staining of the soft tissues (for instance the brain), please, see our publication: https://iopscience.iop.org/article/10.1088/1748-0221/13/02/C02039/pdf

“High-contrast differentiation resolution 3D imaging of rodent brain by X-ray computed microtomography” by Zikmund et al, 2018, JINST.

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