Procedures

Procedures (1)
1. Remove the parts that might cause problems in the clearing and staining (eyes, scales
etc)
2. Also remove guts and gonads
3. Dehydrate in strong ethanol (95%)
4. Acilan blue - stains cartilage (wait 3-24 hours to make sure that it is properly stained)
5. Alizarin red - stains bones, (includes things with calcium)
6. trypsin - breaks down proteins (eats tissue but leaves collagen), also makes it pliable
7. Stored in glycerin - glycerin+collagen has the same bending of light (as it passes
through) = makes it invisible

Procedure (2)
1. Store specimen in 10% buffered formalin
2. Rinse with water
3. Put in 70% ethanol
4. Dye in alcian blue for a day (cartilage)(20mg alcian blue in EtOH (Ethanol) /HAc
(acetic acid) = Solution of 70 mL 100% EtOH: 30 mL glacial acetic acid)
5. Put in plain EtOH/HAc
6. Change to 70% EtOH/HAc and water for a day
7. Change to 1.0% trypsin and 30% saturated sodium borate (should be a day until
the specimen is limp with the blue cartilage shown through
8. Move the specimen to dH2O + 2~5 drops of hydrogen peroxide
9. Transfer to 0.5 KOH (|Potassium hydroxide) (5g of KOH / 1000mL) that has
alizarin red which makes it a very dark purple. Put it in for one day
10. Transfer to 0.5% KOH and change it once or twice over the whole day
11. To make the specimen completely clear, slowly go from 0.5% KOH with glycerin
by ratios of 2:1, 1:1 and 1:2.
12. After that, store in 100% glycerin.
On the clarity of animals: The art and science of clearing and staining. (2013, December 18).
Retrieved October 22, 2018, from http://www.southernfriedscience.com/on-the-clarity-of-
animals-the-art-and-science-of-clearing-and-staining/

Procedure (3)
1. Keep fresh specimens in 10% buffered formalin
2. Take out eyes, get rid of scales etc, anything that will be in the way
3. Put in distilled water for two days (changing water throughout)
4. Soak in a mixture of 10 mL alcian blue, 60 mL of 95% ETOH (ethanol) and 40 mL
glacial acetic acid (cartilage staining)
 5. Put the specimen into 95% ETOH and change once in between 4 hours, then
soak in 70% ETOH for 2 hrs then soak in distilled water (rehydration)
6. Soak in 30 mL saturated sodium borate, 70 mL distilled water, and 1 g of trypsin.
Soak in for 1 to several days depending on when it becomes limp and bones
start becoming visible
7. Put in solution of 100 mL 5% KOH (potassium hydroxide) and 10 mL of alzilian
red for one day (stains the bones red)
8. Store in 75 mL 0.5% KOH, 25 mL glycerin, and 1 mL of H2O2 (hydrogen
peroxide). for a day ~ week
9. Move to 50 mL 0.5% KOH, 50 mL glycerin and 1 mL of H2O2 for same amount
10. Move to 25 mL 0.5% KOH, 75 mL glycerin and 1 mL of H2O2 for same amount
11. Store in glycerin with thymol crystals
Diaphonization / Clearing and Staining Protocol. (n.d.). Retrieved October 31, 2018,
from https://www.scribd.com/document/354718383/Diaphonization-Clearing-and-
Staining-Protocol

Procedure (4)

1. Put the solution in 10% formalin for 2 to 3 days

2. Put in distilled water for 1 or 2 days changing water throughout
3. Put in mixture of 10 mg of Alcian Blue 8GN, 80 mL of ethyl alcohol and 20 mL of glacial
acetic acid for 10 to 12 hours
4. Put in 95% of ethyl alcohol changing 1 or 2 hours
5. Transfer to 75%, 40% and 15% of ethyl alcohol (ethanol) changing 1 or 2 hours in
between
6. Put in distilled water for 2 hours
7. Put in a solution of 30 mL saturated aqueous sodium borate, 70 mL of distilled water and
1g of trypsin
8. Place in 0.5 aqueous KOH solution, which alizarin red dyed in to make it a deep purple-
red for 24 hours
9. Transfer to 0.5% KOH with ¾ KOH and ¼ glycerin, ½ of each KOH and glycerin and ¼
KOH to ¾ glycerin.
10. Finally place it in 100 glycerin to store. With added thymol crystals
Clearing and Staining for bone and cartilage. (n.d.). Retrieved November 1, 2018, from
https://gobiidae.com/methods/method_clear_and_stain.htm