ALIZARIN RED/ALCIAN BLUE WHOLE MOUNT
 Directions are for E17 to E18 day mouse fetuses. Incubation times and volumes must be adjusted for
fetuses of other ages/species or post-natal specimens.
 If the specimen has been fixed in 10% Formalin, use the protocol at the end of this procedure to
prepare the embryos for staining.
Protocol:
1. Sacrifice fetuses. Remove skin, viscera, and adipose tissue from fetuses and place each fetus in a
labeled 20 ml glass vials full of 95% ethanol for 5 days. Agitate occasionally in this and all
subsequent steps.
2. Place specimen in 20 ml 100% acetone for 2 days to remove fat and keep specimen firm.
3. Place specimen in 20 ml Alcian Blue/Alizarin Red Staining Solution @ 37C, for 3 days, up to 7
days.
4. Wash specimen in distilled water.
5. Clear specimen in 20 ml of 1% potassium hydroxide for at least 12-48 hrs; up to 7 days. Change 1%
KOH each day. Proceed to step 6 as soon as skeleton is clearly visible through the surrounding tissue.
6. Continue clearing specimen through 20% Glycerol/1% KOH, ~5 days - 1 week. Progress may be
assessed by transferring the specimen into a petri dish containing 20% Glycerol/1% KOH and
examining it quickly under a dissection microscope. Prolonged exposure to this concentration of
KOH in conjunction with the heat from the microscope bulb causes disintegration of the specimen.
7. Continue clearing specimen through 50% Glycerol/1% KOH, ~5 Days - 1 week.
8. Complete clearing of specimen in 80% Glycerol/1% KOH, ~1 – 2 weeks.
9. Store specimen in 100% Glycerol.
Solutions:
70% Ethanol
100% ethanol
Distilled water
70 ml
30 ml
May be made in advance.
95% Ethanol
100% ethanol
Distilled water
95 ml
5 ml
May be made in advance.
0.3% Alcian blue/70%ETOH Stock:
Alcian blue 8 GX [A3157, Sigma]
70% ETOH
1.5 g
500ml
Updated 04/20/15 by LM
Mix thoroughly and filter into a labeled bottle. May be made in advance and stored at room
temperature.
0.1% Alizarin Red S/95% ETOH Stock:
Alizarin Red S [A5533, Sigma]
95% ETOH
0.5 g
500ml
Mix thoroughly and filter into a labeled bottle. May be made in advance and stored at room
temperature.
Alcian Blue/Alizarin Red Staining Solution:
 can make a master mix; make at least 20 ml/ fetus.
0.3% Alcian Blue Stock
0.1% Alizarin Red Stock
Acetic Acid, Glacial
70% ETOH
1 ml
1 ml
1 ml
17 ml
Make immediately before use.
10% Potassium Hydroxide (KOH) stock:
Potassium Hydroxide
dH2O
100 g
add up to 1 liter
Mix until KOH is dissolved. May be made in advance.
20%Glycerol/1%KOH:
dH2O
Glycerol
10% KOH stock
700 ml
200 ml
100 ml
May be made in advance.
50% Glycerol/1% KOH:
dH2O
Glycerol
10% KOH stock
May be made in advance.
400 ml
500 ml
100 ml
80% Glycerol/1% KOH:
dH2O
Glycerol
10% KOH stock
100 ml
800 ml
100 ml
May be made in advance.
Updated 04/20/15 by LM
Preparation of formalin fixed fetuses for staining:
 Specimens that are originally fixed in formalin will stain less vividly than those fixed initially in
ETOH.
1. Wash specimen in tap water for 24 hours, changing the water every several hours for the first 12
hours.
2. Wash specimens for 2 hours in distilled water, changing the water every 20 minutes.
3. Dehydrate specimen:
a. 50% ETOH, 24 hrs.
b. 70% ETOH, 24 hrs.
c. 80% ETOH, 24 hrs.
d. 95% ETOH, 24 hrs.
 Specimen is now ready for staining.
Reference:
McLeod MJ. Differential staining of cartilage and bone in whole mouse fetuses by alcian blue and
alizarin red S. Teratology. 1980 Dec;22(3):299-301.
Updated 04/20/15 by LM