Mouse Embryo Skeletal Staining (Part 1)

Here is a mouse embryo which has been placed in 100% ethanol. The embryo was euthanized ethically, according to IUCAC protocol.

Mouse embryo

I began to prepare a mouse embryo for a skeletal prep. In order to facilitate diffusion of the stain, I carefully removed some of the tissue around the bone.

Mouse embryo forearm and tendons (left lateral view)

Mouse embryo serratus anterior and arm (right lateral view)

Mouse embryo forearm: biceps, triceps, deltoid (right lateral view)

Mouse embryo forelimb (right lateral view)

In order to visualize cartilage and bone development, 2 staining solutions are used: Alcian Blue and Alizarin Red. 

Alcian Blue will stain cartilage by binding to sulfated glycosaminoglycans. You can shift the specifity of the Alcian Blue by changing the pH, to target a certain type of cartilage. According to Zhao et al., if you want to stain for hyaline cartilage in particular, you should use a pH 1.0 to target the sulfated acidic proteoglycans in the cartilage ECM. (Zhao et al., 2002). Articular cartilage has different properties and hence pH.

Alizarin Red reacts with calcium, resulting in the red staining of bone. Because of this differential binding, it is possible to visualize the process of tissue remodeling in embryos. 

While the embryo represents a frozen snap-shot in time one can see the gradual transition that occurs in the tissue "gradient" as cartilage progressively transitions into bone. Doing a skeletal prep is not necessarily difficult, but creating and optimizing a protocol that works for a new species can be time-consuming.


MAKING THE SOLUTIONS
Alcian Blue

I made a 0.8% Alcian Blue staining solution. For every 100 ml of solution, I added 70 ml of 100% Ethanol with 30 ml of undiluted glacial acetic acid. Then I filtered it through a Nalgene filter. This part can take a long time, as the solute tends to clog the filter quickly. I recommend filling up the filter less than a quarter full, such that you can swirl the container around without spilling. The filtering can go very slow if you don't help the process along.(Note: wear protective gear and dispose of the liquids in a harazardous waste container.)

Filtered Alcian Blue Staining solution

Next, I poured in 20 ml of 0.8% Alcian Blue staining solution. Then I wrapped the tube in foil, to keep light out. I let the mouse embryo sit for 2 days at room temperature.

50 ml conical tube wrapped in foil

Upon inspection, all the tissues of the mouse embryo are dyed a dark blue. Overstaining is a common mistake, as is not letting the prep destain for long enough.

Mouse embryo stained with Alican Blue

Next, I took the embryo through a graded series of ethanol to remove the excess stain; 2 hours each for 75% EtOH - 50% EtOH- 25% EtOH. Then I did 2 washes with distilled water.

Next, I made the Alizarin Red staining solution. Because I was staining an embryo, I didn't use too much KOH to prevent disintegration. I used a 0.1% Alizarin red staining solution and added 0.5% KOH. This will turn the yellowish solution purple. 

I wanted the solution to be a light lavendar purple color. For reference- the solution below is too strong, and should be diluted.

Alizarin Red staining solution

I checked on the embryo regularly to make sure it's not falling apart in the KOH. It remained structurally intact. 

Mouse embryo microscope dissection

Below you can see the mouse embryo on an agar plate, which I use to photograph it. 

Mouse embryo skeletal prep staining with Alican Blue and Alizarin Red

This was my first time doing skeletal prep staining on embryos. I followed a protocol that was optimized for adult lizard skeletons, which called for 2 days of Alcian blue staining, followed by 2 hours each for an ethanol series (100 EtOH - 75 EtOH - 50 EtOH - 25 EtOH) to destain. I was tempted to stain the embryo too heavily, and consequently the skeleton was too dark. I recommend letting the prep destain for 3-6 days for each EtOH step.

I am in my second round of skeletal preps, which include a mouse embryo and 3 chick embryos. This time, I let the preps sit 1 day in Alcian blue staining (at room temp). Then I let them sit overnight in 100% Ethanol to destain. After that, I will let them sit for 3 hours each in 75 EtOH - 50 EtOH - 25%  EtOH to destain. 

The result should be a lighter stain, which allows me to visualize the Alziarin red (bone) staining better. In particular, the matrix transitions from bone to cartilage should be easier to see. Regardless, below is my first try at a mouse skeletal prep (after 2 days of Alcian blue staining and 4 hours of Alizarin red staining).

Right radius and ulna of a mouse embryo skeletal prep (right lateral view)

Mouse embryo forelimb: humerus, radius and ulna (right lateral view)

Below is the scapula, arm and back.

Mouse embryo thoracic and shoulder girdle (right lateral view)

Mouse embryo skeletal prep:os coxae, femur, tibia and fibula (right lateral view)

Below is the lower leg of the mouse embryo. 

Right hindlimb of the mouse embryo: tibia, fibula and foot metatarsals (right lateral view)

 
AFTER 18 HOURS OF ALIZARIN RED

Mouse embryo skeletal prep staining with Alizarin Red: os coxae, femur, tibia and fibula (right lateral view)

Below are the vertebral rings, with the initial signs of Alizarin red (calcium) staining.

Developing vertebrae of a mouse embryo (lateral view)

In the ribs, you can clearly see where the bone and cartilage transition occurs. 

Mouse embryo ribs and costal cartilage (right lateral view)

The mouse embryo's skull bones are visisble below. The Parietal (top most) part of the skull is bright pink.

Skull bones:temporal, parietal and frontal bones; C1 atlas vertebrae of a mouse embryo (right lateral view)

Below are the metatarsals and phalanges of the foot. There is an interesting pattern below, as it appears the toes are mostly cartilage (blue) with strips of calcified matrix in the middle of the metatarsals (I think).

Mouse embryo foot with metarsals (right lateral view)

Mouse embryo skeletal prep: humerus, radius and ulna (right lateral view)

Mouse embryo skeletal prep: forelimb (left lateral view)

2 DAYS AFTER ALIZARIN STAINING

Mouse embryo humerus, radius and ulna (left lateral view)

Mouse embryo skeletal prep (left lateral view)

Hindlimb of a mouse embryo skeletal prep (left lateral view)

Below is the vertebral column near the hip joint. You can see one of the coxal bones on the side.

Lumbar vertebrae of a mouse embryo skeletal prep (left lateral view)

Developing vertebrae of a mouse embryo

Next I began to put the embryo through a 1:4 ratio of 1% KOH to Glycerol for 1 day, then a 1:1 ratio of 1% KOH to Glycerol, followed by a 4:1 ratio of 1% KOH to Glycerol. I store the embryo in 100% glycerol. 

Below you can see the embryo skeletal prep floating in a 1:1 solution of 1% KOH to Glycerol.

Mouse embryo skeletal prep in a KOH:Glycerol solution

For the second round of mouse skeletal preps, I was able to destain the embryo a bit more, though I still have not reached the optimal point of staining yet. 

Mouse embryo skeletal prep neck and skull (let lateral view)

Below is the knee, with the femur to the right and the tibia and fibula to the left. There is an odd "ball" of cartilage on the inside of the knee. 
 

Left Hindlimb: femur, patella, tibia and fibula of a mouse embryo (left lateral view)

References
Zhao, Qiuong. et al., (2006).  Expression of parathyroid hormone-related peptide (PTHrP) and its receptor (PTH1R) during the histogenesis of cartilage and bone in the chicken mandibular process. J Anat. 2002 August; 201(2): 137–151.


All embryos were treated ethically according to IUCAC protocol.




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