Dear Alexander,

Below is the protocol we are routinely using in the lab.

Please inspect daily how quick is the dissolution of soft tissue. Also it is a basic protocol keeping in mind that E19.5 is almost a newborn... so please adapt it and leaving organs in embryo is mainely for mid-gestation embryo such as E13.5-E15.5 then taking out liver is better. I don't know exactly Acetic acid is doing my guess will be that it heps either ARS or Alcian to be better dissolved in alchoolic solution.

Wish you good luck with your experiment.

Best

Daniel

From Newborn mice :

• Eviscerate mice and take apart piece of internal organs for genotyping (liver)

• Remove skin

• Corpses are fixed for 5 days in Ethanol 95%

• Acetone for 2 days

• Stained in 0,005% Alizarin red S

0,015% Alcian Blue GS

in 5% Acetic Acid, 5% H2O and 90% Ethanol for 3 days at 37C

• samples are washed in H2O and cleared in KOH 1% for 48 hours

• 0,8% KOH , 20% glycerol 1 Week

• 0,5% KOH , 50% glycerol 1 Week

• 0,2% KOH , 80% glycerol 1 Week

• 100% Glycerol for storage

Cartilage is stained blue

Bone is stained red (calcium containing tissue).

From embryo :

Same protocole but don´t remove skin and organs.

Staining for 5 days instead of 3 days and clearing is shortened

Hi,

There is no efficient fast way... the balance is delicate between KOH mediated tissue digestion and keeping the skeleton in one piece. You have to be aware that at E15.5, the skeleton could be easily broken during the dissolving step, addition of glycerol make the dissolution more sublte but slow down the process.

I am not sure I have been overy helpful, but I recommand you not to be too fast. More quicker is a µ-CT analysis :-)

Daniel

Thanks Daniel,

Some days ago i tried this protocol on E 15.5 embryos. Actually, i was not able to properly skin the embryos, neither by boiling them in 70°C for 30sec nor after incubating them in 4% salt water O/N at 4°C. the resulting embryos were only slightly stained for cartilage, but it was odd that the maceration by KOH didn't worked - the embryos still look pale and not clear.

Interestingly, I also tried out a method discribed here (http://www.ncbi.nlm.nih.gov/pubmed/17822689). As it turned out the staining was more prominent and the embryos were easier to clear in KOH/Glycerol then with the other method.

I have one more question: what exactly does glacial acetic acid? Because it is the only chemical which is used in the 2nd method but not in the 1st one.

in my experience, skinning is not anymore necessary to stain E15.5 embryos, some protocols would use a trypsin step depending on the age the samples. The hydration and dehydration step, as well as KOH steps are critical to allow proper staining of your tissue samples... I usually use low concentrations of KOH to allow longer time of Alizarin red staining and clearing skin, muscles and visceral tissues without dissolving the tissues.

Dear Alexander,

Below is the protocol we are routinely using in the lab.

Please inspect daily how quick is the dissolution of soft tissue. Also it is a basic protocol keeping in mind that E19.5 is almost a newborn... so please adapt it and leaving organs in embryo is mainely for mid-gestation embryo such as E13.5-E15.5 then taking out liver is better. I don't know exactly Acetic acid is doing my guess will be that it heps either ARS or Alcian to be better dissolved in alchoolic solution.

Wish you good luck with your experiment.

Best

Daniel

From Newborn mice :

• Eviscerate mice and take apart piece of internal organs for genotyping (liver)

• Remove skin

• Corpses are fixed for 5 days in Ethanol 95%

• Acetone for 2 days

• Stained in 0,005% Alizarin red S

0,015% Alcian Blue GS

in 5% Acetic Acid, 5% H2O and 90% Ethanol for 3 days at 37C

• samples are washed in H2O and cleared in KOH 1% for 48 hours

• 0,8% KOH , 20% glycerol 1 Week

• 0,5% KOH , 50% glycerol 1 Week

• 0,2% KOH , 80% glycerol 1 Week

• 100% Glycerol for storage

Cartilage is stained blue

Bone is stained red (calcium containing tissue).

From embryo :

Same protocole but don´t remove skin and organs.

Staining for 5 days instead of 3 days and clearing is shortened

Thanks Daniel and Dennis,

In a few days I will be preparing E18.5 embryos, which are hopefully easier to skin. The E 15.5 embryos were easy to eviscerate btw, since the liver is taking up most of the viscera space.

All in all my first try with the E15.5 mice and your input here convinced me that the so-called "fast" staining protocols are a wishful thinking and one has to adjust carefully and patiently all the fixation,staining and clearing steps there are.

I will try your protocol next time and will give some feedback here afterwards.

And btw: I also had the idea with the micro-CT, i will try that one too.

Hi again,

I wanted to comment my recent attempt to make a whole mount double staining for E18.5 mouse embryos.

First of all, there was a big difference when i tried to skin the E15.5 and now the E18.5 offspring. Where the former ones turned out to be too fragile, the latter embryous were easy enough to skin after some hours of softening in water and 1 min aditional boiling. This was important, because alcian blue won't penetrate the skin of older embryos (at least by my approach).

I have attached a picture of one E18.5 double-stained mouse. The cartilage staining is still a bit to strong which led to a high backgroud (unspecific tissue-staining).

Thank you all again for the clues.

Hi Alex,

Great ! As you could see in my previous post, indeed you don't have to skin E15.5, the dye will penetrate, while E18.5 are newborn alike. you could rid off the background by stepwise going from KOH 1%, 0% gycerol to KOH, 0%, glycerol 100% then you get a very nice staining. Alizarin looks great on your prep !

Daniel

Thanks,

As you posted earlier, it's delicate to keep the skeleton integrity intact during the soft tissue maceration. After the EtOH-fixation and cartilage staining I started with a 2% KOH digestion for several hours after which i switched to a 1,5% KOH Alizarin solution for 3h and an additional 1% KOH digestion O/N. the next day the E18.5 were already clear (as seen on the picture), so the KOH-solution was changed for a 25% Glycerol (in H2O). They are now equilibrated in 50% Glycerol.

Now, is there a chance to further clear the tissue by applying low % KOH-solutions again to the Embryos? My concerns are that this could damage the embryos more than it could clear them any further.

Can someone can help how to take the photos of the mice embryo.. just after taking and before fixation..

Dear Irfan

keep them in solution best is 100% glycerol

Best

daniel

Dear Daniel, Thanks for your suggestions. still one question..

Is Glycerol interfere with the fixation??? I want to fix in 4% PFA for the sectioning.. Thanks

/Irfan

Hi Irfan,

I am not sur to get it right but the above protocol is for whole mount staining.

there are couple of caveats if you want to section the stained skeleton, and it depends also how you will proceed with fixation and decalcification (or not plastic section vs paraffin or frozen). Just to remind you Alzarin stains calcium so it is sensitive to decalcification procedure

Best

Irfan, I think that 4% PFA wouldn't interfer to much with the cleared embryo - but you might find that PBS makes the sample a bit cloudy again.. so ideally prevent that.

Also, for taking the pictures you can use any macro-photography setup or a standard stereo microscope.

Hi,

Do you change the medium every day when you put embryos in solution for few days ? 

Thank you in advance for your answer

Hi,

Since I have already fixed the embryo in 100% ETOH , may  I boiling the embryo after fixed? I wanna get a clear toes, so does anyone have any good ideas  how to remove the skin as much as possible?

Marie, change the medium as often as possible (more frequently in the begining).

Morgan, I am not boiling the embryo, warmer is the KOH solution more strong will be the effect. With embryo it is much more safe to go slowly.

for the skin depends on the embryo stage, but be gentle with forceps.

Hope this help

Best

Daniel

Hi, I want to make skeletal preps of newborn mice. Can anyone please share there protocol.

Thanks