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1
Preface
Diaphonization (also known as clearing and staining) is a preservation technique in which the tissues
of a specimen is made transparent, and the cartilage and bones are made visible by use of specific
dyes.
Diaphonization is nothing new – in fact it has been around since before the 1940’ies, where the
process enabled zoologists to study the bones while still inside the animal. The flexibility of the
specimens even allowed them to study these bones in movement, and it was therefore a powerful
tool in order to better understand bone function. While this technique is still used in zoology, there
has been a recent spark of interest in diaphonization outside the zoology field due to their visual
appeal. Diaphonization is now also part of the taxidermy community, and diaphonized specimens are
being sold as artsy display items worldwide. Part of this comes down to the simplicity of the
technique; diaphonization is a pretty easy and forgiving process that is possible to do with relatively
few remedies and without advanced lab equipment. This means that – in theory – this could be done
in the comfort of your own home.
While there are a lot of protocols out there, they can be hard to find and navigate without a scientific
background. The idea of this protocol is therefore to help bring diaphonization to the layman.
About me: I have a background in science (biochemistry) and spend a lot of my time in a lab. I started
diaphonization as something fun to do at home, and thought I would share it with the world. While I
do have experience in general lab-related tissue/cell dyeing, diaphonization is still new to me, and I
am in no way a professional. If you find something gravely wrong, or anything else that could be
improved, please don’t hesitate to contact me J
Table of contents
Introduction 3
Materials 3
A ‘quick’ note on safety 4
Calculations 5
F.A.Q. 6
References 6
Protocol: Experimental overview 7
Part 01: Preparing the specimen/skinning 8
Part 02: Fixation 9
Part 03: Washing 10
Part 04: Cartilage staining (Alcian Blue) 11
Part 05: Rehydration 12
Part 06: Trypsin digestion 13
Part 07: Bone staining (Alizarin Red) 14
Part 08: Clearing 15
Part 09: Results 16
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Introduction
This is the boring part, but it needs to be here. It has a list of the materials needed, but it also includes
some words about safety when working with these types of chemicals, and a section on how to do
some typical lab work calculations that you might need during this protocol.
Materials
LIST OF CHEMICALS NEEDED:
Item Notes
This protocol is mostly suited for small vertebrates like mice –
Specimens some animals may require tweaking of the protocol in order to
work. Frozen feeder animals are good to start with.
Surgical kit for dissecting Get at least 1 decent pair of scissors + forceps
Graduated cylinders A set of e.g. 100 mL, 20 mL and 5 mL will get you a long way
Scale + weighing paper + spatula You want a scale that is able to weigh down to 0.01g
These are used throughout the protocol, so you’ll want a good
Jars/containers amount of these in various sizes. Old food jars are fine as long
as they are cleaned well
Safety You want gloves, a mask and some googles
Get some containers for your waste liquids, so that you can
Waste containers
dispose of this properly (check your local waste rules)
3
A ‘quick’ note on safety
Some of these chemicals are quite nasty – both for the body and the environment. It is always a good
idea to know your chemicals before you start working with them. Detailed safety sheets should come
with the chemicals when you buy them, but here is a quick summary:
GHS explanations:
DILUTION aka. “Help! I have solution of X%, but I need a concentration of Y%”:
The answer is to make a dilution. Let’s say we have bought some formalin that is 23.4%, but we need
to make a 10% dilution, and we want 150 ml of it. In this case we need to do some calculations.
$% ∙'%
The old-school method is to do manual calculations using the formula: 𝑉" = , where
$(
C1 = concentration of stock, V1 = volume to take out from the stock, C2 = final
concentration, V2 = final volume.
So if I have a stock of 23.4% (C1) formalin, and I wish to make 150 ml (V2) of a
$% ∙'% ")% ∙ ",) -.
10% (C2) formalin solution, I will need to take out: 𝑉" = = = 64 𝑚𝑙
$( /0.2%
from my stock solution, and add up to 150 ml with distilled water, which comes
to 𝑉/ − 𝑉" = 150 𝑚𝑙 − 64 𝑚𝑙 = 86 𝑚𝑙 distilled water (see figure to the right)
The easy method is to use a dilution calculator, which does exactly the same thing. A good one can
be found here: http://www.physiologyweb.com/calculators/dilution_calculator_molarity_percent.html
Plot in the numbers of your stock concentration
(here 23.4%), the final concentration (here
10%), and the final solution volume (here 150
ml), and click calculate. The calculator will give
the answer in yellow.
The tool is useful in several ways. Say you have 1L of the 23.4% formalin stock, and you wanted to
dilute everything at once, you could leave the “final solution volume” field to calculate the amount of
water needed. So if I wanted to dilute my entire 1L bottle, I would need to fill up to 2.34L with water (so
a total of 2.34 − 1𝐿 = 1.34𝐿 water)
(*Now, technically 1 ml = 1 g is a golden standard that doesn’t apply to all chemicals, and to be absolutely correct
you would need to calculate the mass per volume using the molar mass of the specific compound to get the right
amount to add. However, when working in the lab scientist like to be lazy – very lazy, so lab protocols are most
often written with that in mind – as will this)
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F.A.Q.
Which animals can I use?
This is a question of trial and error. So far the technique has been successful on birds, mammals,
amphibians, reptiles and fish. A general rule is: The larger/more dense the specimen, the harder to
clear. Small vertebrates therefore give the best results. Remember that this protocol is optimized to
small vertebrates like rodents and birds, and that some of the steps may vary a bit between species.
Feather, fur and scales all needs to be removed in order to get a successful clearing.
References
PRIMARY SOURCES:
“Dyeing The Dead” YouTube series by TacoKel: A very nice Diaphonization series on YouTube, where
https://www.youtube.com/user/TacoKellz she shows the entire process. Her videos are more
talkative than mine, and she does a great job at
explaining everything in an easy to understand language.
Taylor, W. R. (1967). An Enzyme Method of Clearing Very thorough protocol. A bit long for daily use, but good
and Staining Small Vertebrates. Proceedings of if you are troubleshooting, or to get a nice introduction to
the United States National Museum, 122(3596), diaphonization
1–17.
Weck, B., & Miljak, P. (1998). Give New Life to Old If you want a “real” protocol to read this is the one. Simple
Specimens through Clearing & Staining. The and easy to follow. The bad news is that it requires a
American Biology Teacher, 60(9), 699–702. subscription and is therefore not freely available for most
people
Cortés-Delgado, N., Pérez-Torres, J., & Hoyos, J. M. Not as nice at the Weck & Miljak one, but still easy to
(2009). Staining Procedure of Cartilage and read. This one is much more easy to find, as it is freely
Skeleton in Adult Bats and Rodents. International available online
Journal of Morphology, 27(4), 1163–1167.
SECONDARY SOURCES:
Green, M. C. (1952). a Rapid Method for Clearing and Staining Specimens for the Demonstration of Bone. The Ohio
Journal of Science, 52(1), 31–33.
Tipton, P. W., & Burtt, M. E. (1977). A method for mechanised staining of rat and mouse foetuses for teratological
examination. Laboratory Animals, (11), 265–267.
Dingerkus, G., & Uhler, L. D. (1977). Enzyme clearing of alcian blue stained whole small vertebrates for
demonstration of cartilage. Stain Technology, 52(4), 229–232.
Taylor, W., & Van Dyke, G. C. (1985). Revised procedures for staining and clearing small fishes and other
vertebrates for bone and cartilage study. Cybium.
Armbruster, J. W. (1989). Clearing and Staining Methods. Journal of Chemical Information and Modeling (Vol. 53).
6
Protocol: Experimental overview
STEP 1: Prepare, fix and wash
10 mg Alcian Blue
60 ml 95% Ethanol
40 ml Glacial acetic acid
1 day
STEP 3: Rehydration
1 day
TUTORIAL:
The method of skinning is very much up to personal preference. There is no perfect way of doing this.
The most important thing is to not disrupt any bones in the process. This is how I do it (using a frozen
feeder mouse as an example):
VIDEO TUTORIALS:
Skinning of mouse: https://youtu.be/5eO1DkXtUCM
Skinning of chicken: https://youtu.be/b6OfTxtizrU
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Part 02: Fixation
To preserve the specimens and stop them from decaying they are fixed in formaldehyde. After this
step the body will become rigid and slightly dull in color.
TUTORIAL:
After skinning and removal of fat and organs, the specimen needs to be fixed in formalin.
1) Make a 10% formalin solution and pour it into a jar with a lid. There should be enough solution to
cover the specimen(s) entirely (usually 100-200 ml in a glass jar is sufficient for small animals)
2) Transfer the specimens into the formalin solution, making sure they are completely covered and
close lid.
3) Leave in solution for at least 2-3 days (this time frame is fit for small animals like mice, but may
vary for other species). If you need to take a break in the protocol, this is a good time to do so, as
the time in formalin is not that critical. In fact the animals can be stored in formalin for years if
needed (Weck & Miljak, 1998).
VIDEO TUTORIAL:
https://youtu.be/zmJp7kQem10
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Part 03: Washing
After fixation the specimen needs to be washed in order to rehydrate the tissues and to remove
excess formaldehyde. This is done through a series of water baths over several days.
TUTORIAL:
The specimen needs to go through a series of water baths. The baths are as following:
Day 1: Distilled water, leave overnight
Day 2: Distilled water, leave overnight
Figure 6: During water baths. The specimens are stiff and colorless after fixation.
VIDEO GUIDE:
https://youtu.be/B1-VyqGwqjU
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Part 04: Cartilage staining (Alcian Blue)
This is the fun part! In this step the cartilage will be stained blue using the dye Alcian Blue.
TUTORIAL:
For the staining we need to make the dye solution:
1) Measure out an appropriate amount of Alcian Blue powder using a scale. The easiest way is to
use weighing paper and scoop the product out with a spatula (Figure 7). For 200 ml you’ll need to
measure out 20 mg (= 0.02 g) Alcian Blue (See Table).
2) Pour the Alcian Blue powder into an empty jar.
3) Add the 95% Ethanol to the jar (see Table for amount)
4) Add the glacial Acetic Acid to the jar (See Table for amount)
5) Put lid on and mix by shaking gently
6) Transfer the specimen to the jar with the dye mix, and make sure that it is completely covered.
7) Let it soak in the dye for 1 day before moving on to rehydration…
→ The dye solution can be used more than once. Stop using if build-up starts to appear at the
bottom.
VIDEO TUTORIAL:
https://youtu.be/SIAypcts298
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Part 05: Rehydration
Here the specimen gets rehydrated and neutralized after the acetic cartilage staining by going through
a series of baths of ethanol and water.
TUTORIAL:
The specimen needs to go through a series of baths. The baths are as following:
BATH I: 95% ethanol, 2 hours - 1 day
BATH II: 95% ethanol, 2 hours - 1 day
BATH III: 70% ethanol, 2 hours - 1 day
BATH IV: Distilled water, 2 hours - 1 day
Day 1, Bath I:
1) Transfer specimen to a new jar and pour over 95% ethanol until specimen is covered. Let sit
overnight
VIDEO TUTORIAL:
https://youtu.be/K-tIGuw2jIs
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Part 06: Trypsin digestion
Trypsin is a digestive enzyme that breaks down many proteins, but leaves collagen. The breaking
down of muscle and other tissues will help to make the animal transparent, while the collagen will keep
the specimen from falling apart.
TUTORIAL:
1) Since Trypsin works best at a slightly elevated pH (~7.5-8.5), an alkaline buffer of sodium borate
(borax) is made:
a) Boil some distilled water (you’ll need 60 ml for a 200 ml solution)
b) Transfer the hot water to a heatsafe container and add borax until saturated. It is saturated
when small insoluble crystals form at the bottom.
c) Trypsin is temperature sensitive, so cool to room temperature before moving on.
2) Once the borax buffer has cooled completely, you’ll need to make the digestion mixture:
Digestion mixture
Factor For a 200 ml solution:
Borax buffer solution 30% 60 ml
Distilled water 70% 140 ml
Trypsin 1:100 2g
Total 200 ml
3) Measure out the borax buffer, water and trypsin (see table for volumens) into an empty jar.
4) Put on lid and shake gently to mix.
5) Transfer specimens to the digestion mixture
6) The incubation time will depend on size and build of the specimen. Check on the specimens daily
to look for signs of proper digestion. You want the specimen to be limp and bones to be slightly
visible (Figure 10 + Figure 11) before moving on to the next step.
7) Continue immediately to bone staining once ready…
→ Usually by 2-3 days with small animals like mice. Change the solution after 3 days if longer
incubation is needed.
Figure 10: Bone visibility in a mouse after 3 days of digestion. Figure 11: Bone visibility in a mouse after 3
Ribcage is clearly visible. days of digestion. Bones in hind legs are
slightly visible (yellow arrow)
VIDEO TUTORIAL:
https://youtu.be/Y_5HTxB0kXM
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Part 07: Bone staining (Alizarin Red)
This is the second dye step. Here the bones will be stained that characteristic purply red color using a
dye called Alizarin Red.
TUTORIAL:
Dye recipe
Factor: For a 250 ml solution: *Dependent on specimen size and density.
KOH 0.5-2%* 1.25-5 g 0.5% is fine for small animals like mice
Alizarin Red S 1:10,000 0.025 g
Distilled water 250 ml
Total 250 ml
1) Measure out the KOH needed (see Table) and add to a new jar
2) Add distilled water
3) Measure out the Alizarin Red S (you only need a tiny amount) and add to the jar (Figure 12)
4) Put on lid and shake to mix
5) Transfer the specimens to the dye solution and stain for 1 day before moving on to clearing…
→ The dye solution can be reused as long as there is no precipitation.
Figure 12: Ooh, pretty Figure 13: Specimens in Alizarin dye solution
VIDEO TUTORIAL:
https://youtu.be/YhxgNgd1OMQ
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Part 08: Clearing
This is the longest part of the protocol, but also the part where we see the biggest changes. The
specimen will go through a series of baths to make it transparent. The longer the time spent in these
baths, the better the clearing – however be careful not to leave specimens for too long as this might
make them come apart.
TUTORIAL:
The specimen needs to go through a series of clearing baths. The baths are as following:
BATH I: 3:1 0.5% KOH to Glycerin (Optional: 1:100 3% H2O2), leave 1 day to over a week
BATH II: 1:1 0.5% KOH to Glycerin (Optional: 1:100 3% H2O2), leave 1 day to over a week
BATH III: 1:3 0.5% KOH to Glycerin, leave 1 day to over a week
1) Start by making a 0.5% KOH stock solution. Make enough for all 3 baths:
0.5-2% KOH stock solution
Factor For a 300 ml solution: **Dependent on specimen size and
density, 0.5% is fine for small animals
KOH 0.5-2%** 1.25-5 g like mice, but 2% is better for larger
Distilled water 250 ml and/or more dense animals
Total 250 ml
3) Transfer the specimens to bath I and incubate for 1 day to over a week
4) Transfer the specimens to bath II and incubate for 1 day to over a week
5) Transfer the specimens to bath III and incubate for 1 day to over a week
VIDEO TUTORIAL:
https://youtu.be/EVQ28494ymc
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Part 09: Results
Now it’s time to finish up the specimens. The specimens should at this point be quite transparent with
visible staining. The last bit of transparency we’ll get by the use of magic! Or well, basic science.
Remember how trypsin digested everything but collagen? Collagen and glycerin has the same
refractive index, meaning they bend light in the same manner. When light can pass freely through two
objects without bending they will appear transparent. That means that placing the collagen-rich
specimen into 100% glycerin will give the illusion that it´s even more transparent. Neat, right?
TUTORIAL:
1) Take the specimen out of the jar for inspection
2) Cut off any loose tissue or small imperfections that doesn’t look good (Figure 15)
3) Once satisfied put the specimen into a jar that fits the specimen nicely
4) Fill the jar with glycerin
5) Add a pinch of thymol crystals – this will work as an anti-fungal agent
6) Use forceps to play around with the placement of the specimen in the jar, add more glycerin if
needed, and put the lid on once satisfied. Sometimes the specimen can be a little floaty and hard
to work with. This is due to tiny air bubbles stuck inside cavities - don’t worry, they will disappear
over time. Just leave the specimen for a few days/weeks to allow the bubbles to clear, and then
try to reposition it again.
7) You are done! Go enjoy your morbidly cool looking creatures!
Figure 15: Removing excess tissue Figure 16: The final product! If you were not careful
during the skinning process it will show (hence the
broken leg)
VIDEO TUTORIAL:
https://youtu.be/fH_5GvM8UdI
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