Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The overall goal of the following experiment is to effectively assess sensory axon growth phenotypes in developing fetal mice. This is achieved by first properly breeding and genotyping tre a tau laxy reporter mice such that downstream staining assays can be performed as a second step. The mouse embryos are fixed and stained to allow for visualization of sensory axon projections in the whole animal.

Next, the embryos are dehydrated and cleared in order to completely visualize the deep lying axon projections that would otherwise not be visible. The results demonstrate that these methods are an effective means of rapidly assessing embryonic nociceptive axon growth phenotypes. Using the track a Tau Lae reporter, This method can help answer key questions in the field of sensory axon growth by allowing researchers to cross their gene of interest onto the track a tlac Z background and screen for any changes to sensory axon growth phenotypes.

The implications of our technique extend toward therapy in the sense that scientists can use our technique to test whether known congenital mutations may cause sensory axon growth phenotypes. To begin euthanized timed pregnancy females by cervical dislocation as referenced in the text protocol, proceed to dissect embryonic E 16 to E 18 embryos from timed pregnancy females then place the embryos individually into the wells of a six well dish filled with cold phosphate buffered saline or PBS. After rinsing the embryos in cold PBS remove a small portion of the amniotic membrane of the embryo and place it in a prepared proteinase K solution from the DNA purification kit or subsequent genotyping.

Alternatively, if the amniotic membrane is lost, remove a small portion of the tip of the embryo tail and place it in proteinase K solution. Next, poke into the skin all around the embryo using insect pins making about 100 holes per side. Remove the PBS and slowly add fresh 2%paraform aldehyde or PFA pH 7.4 to the well after gently shaking for two hours at four degrees Celsius.

Rinse embryos twice in PBS and store in PBS at four degrees Celsius until staining For genotyping. Immerse the amniotic membranes or embryo tails in the proteinase K mixture according to the manufacturer's instructions. Incubate at 56 degrees Celsius for 10 minutes.

After extracting the DNA using a commercially available DNA purification kit, use 0.5 microliters of the total 200 microliters of purified genomic DNA solution to prepare a PCR reaction as described in the text protocol, then proceed to run the PCR program as detailed there. Following PCR, run the PCR samples on a 2%aros gel at 100 volts in one extras acetate EDTA buffer for 20 minutes. Include a lane holding a DNA ladder to assess fragment lengths and stain the gel using routine protocols.

Analyze embryos for the presence of wild type, track a, track a tau lae and back null alleles. To stain the embryos. Use a commercial kit for lax e staining with the modifications described here.

To begin immerse embryos in buffer A for at least one hour shaking gently at room temperature. Then directly immerse embryos in buffer B for at least 15 minutes, shaking gently at room temperature. Next, dilute a 40 milligram per milliliter xal stock solution into prewarm tissue based solution at a concentration of one milligram per milliliter.

And add five milliliters of the resulting mixture to a glass scintillation vial. Transfer the embryos to the glass scintillation vial containing the xal tissue based mixture. Seal the lid with perfil and incubate at 37 degrees Celsius overnight.

Checking for the presence of blue stain. Then postfix the embryos with fresh 4%PFA pH 7.4. Gently shake for four hours at four degrees Celsius before rinsing the embryos twice in cold PBS to dehydrate the tissue place embryos in 50%methanol 50%PBS mixture for 30 minutes at room temperature shaking in the glass.

Continue to dehydrate at room temperature shaking in glass files in 75%methanol for 30 minutes, followed by 90%methanol for 30 minutes. Finally, dehydrate twice in 100%methanol for 30 minutes. Then immerse the embryos in a one-to-one mixture of 100%methanol and 100%benzoyl alcohol and benzoyl benzoate or BABB for 15 minutes.

Make sure to use glass because the BABB will dissolve plastic as a final step. Immerse embryos in 100%BABB to completely clear the tissue and to visualize X cal staining in the cleared embryo or mount imaging, stabilize the embryo immersed in 100%BABB at the appropriate angles for photography using metal forceps if necessary. Illuminate using a combination of uplight and under light.

Light sources. Acquire images with a dissecting microscope outfitted with a digital camera. Take multiple brightfield exposures at five successive focal planes.

0.5 millimeters apart along the Z axis. Exposure times and F-stops may vary according to illumination and camera specifications and need to be optimized for each setup. Use standard image processing software to process overlay images and generate photo montages.

The genotypes of track a tal Lai homozygotes back null and track a tal lai heterozygotes back. Snu embryos can be unambiguously determined by standard PCR genotyping. ALS staining displays detailed subcutaneous peripheral axonal arbors in the embryos axon projections from trigeminal ganglia and torso and head are shown in the uncleared embryo.

Here, ex GS staining displays detailed peripheral axonal arbors in projections from dorsal root ganglia in mid torso before and after clearing with BABB in embryos that completely lack Track A signaling but carry the constitutively kinase activated. BRAF mutation under control of the neuron specific nest. In promoter, the morphology of nociceptor peripheral projections developed near normally.

This demonstrates a crucial function for BRAF signaling in peripheral axon growth and shows that the survival and axon growth promoting functions of track A signaling can be separated with the back null genetic background substituting for the loss of track A dependent survival signaling. It is important not to over fix the embryos, which could compromise the enzymatic activity of beta glaces. Also remember that BABB is a very hazardous material and when working with it, you should always use proper ventilation and wear proper personal protective equipment.

After watching this video, you should have a good understanding of how to effectively process, stain, and visualize laxy stained axons in the intact animal.