Alcian Blue – Alizarin Red Staining of Mouse Skeleton   

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In this protocol


Our lab has used the Alcian blue – Alizarin red staining method with certain modifications to characterize skeleton deformities in mice lacking Pek/Perk, encoding a translational control eIF2alpha kinase.

Materials and Reagents

  1. Neural buffered formalin (Sigma-Aldrich, catalog number: HT5014 )
  2. Alcian blue 8GX (Sigma-Aldrich, catalog number: A5268 )
  3. Trypsin (Sigma-Aldrich, catalog number: T1426 )
  4. Saturated sodium borate (Sigma-Aldrich, catalog number: S9640 )
  5. Alizarin red (Sigma-Aldrich, catalog number: A3882 )
  6. Thymol (EM Life Science, catalog number: TX0615-1 )
  7. Ethanol
  8. Glacial acetic acid
  9. Potassium hydroxide
  10. Glycerol
  11. KOH
  12. Trypsin solution (see Recipes)


  1. Conical tube


  1. Adults:
    1. Fix mouse skeleton in 10% neutral buffered formalin for at least 24 h.
    2. Rinse the sample in ddH2O O/N (1 h for embryos) with gentle shaking, and post-fix it in 70% ethanol.
      Note: At this point, samples can be stored in 70% ethanol for a long period.
    3. Remove skins and internal organs carefully from the sample.
      Note: Remove all skins, even those on the small toes.  
    4. Stain the sample with 0.02% Alcian blue 8GX (prepared in ethanol/ glacial acetic acid, 7:3) for 1 to 2 days.
      Note: Cartilage tissues will be stained blue.  
    5. Wash the sample with plain ethanol/ glacial acetic acid (7:3) for 1 h.  
    6. Soak the sample in 100% ethanol O/N, and then in ddH2O for 1 to 2 days.  
    7. Treat the sample with 1.0% trypsin (prepared in water solution containing 30% saturated sodium borate) O/N.
    8. Should limp and blue cartilage be readily observed at this point, proceed to stain the sample with Alizarin red (prepared in 0.5% KOH) O/N.
      Note: Add enough (no specific amount) saturated Alizarin red until the solution appears dark purple. Mineralized bones will be stained red.  
    9. Treat the sample with a gradient series of 0.5% KOH/ glycerol (i.e., 2:1, 1:1, 1:2 and 100% glycerol, 2 days for each step), and store it in glycerol with a crystal of thymol.

  2. Embryos:
    1. A similar protocol can be used to stain embryos. To do this, fix embryos in 90% ethanol for at least 1 week.
    2. Treat the sample with 0.01% Alcian blue 8GX for 3 days, and then perform rehydration through a gradient series of ethanol (70% ethanol, 2 to 3 h, twice; 40% ethanol, 2 to 3 h; 15% ethanol, 2 to 3 h; ddH2O, until the sample sinks to the bottom of a conical tube). Treat the sample further with fresh 1% KOH for 1 to 2 days until it becomes clear.  
    3. Treat the sample with 0.001% Alizarin red for 2 to 3 days until the bone becomes purple.
    4. Rinse the sample ~ 3 times in 1% KOH, several hours each time.  
    5. Treat the sample through a gradient series of glycerol-KOH (20% glycerol/ 1% KOH, 24 h; 50% glycerol/ 1% KOH, 24 h; 80% glycerol/ 1% KOH, 24 h; 100% glycerol, 24 h x 2).

Representative data

Figure 1. This figure is adapted from the original (Zhang et al., 2002).
Shown here is Alizarin Red (mineralized bone) and Alcian Blue (cartilage) skeletal staining of 18-day-old wild-type A and Perk-/- mutant mice B. The mineralization of the flat bones of the skull (P, parietal; O, occipital; T, temporal) is greatly reduced in the Perk-/- mutant mouse.


  1. Trypsin solution
    1.0% trypsin
    30% saturated sodium borate


This protocol was adapted from previously described work by Hanken and Wassersug (Hanken and Wassersug, 1981). PZ was supported by a research assistantship in the Cavener lab at the Pennsylvania State University. This work was supported by an NIH R01 grant awarded to DC.


  1. Hanken, J., Wassersug, R. J. (1981). The visible skeleton. Funct Photogr 16(4): 22-26, 44.
  2. Zhang, P., McGrath, B., Li, S., Frank, A., Zambito, F., Reinert, J., Gannon, M., Ma, K., McNaughton, K. and Cavener, D. R. (2002). The PERK eukaryotic initiation factor 2 alpha kinase is required for the development of the skeletal system, postnatal growth, and the function and viability of the pancreas. Mol Cell Biol 22(11): 3864-3874.
Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Zhang, P. (2012). Alcian Blue – Alizarin Red Staining of Mouse Skeleton. Bio-protocol 2(8): e162. DOI: 10.21769/BioProtoc.162.

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farzane sadeghi
tehran university
can we remove skin and internal organ at first and then fix the samples?
3/3/2014 8:26:32 AM Reply
farzane sadeghi
tehran university
I appreciate from your protocol, your assistant and your guide.
I did yor protocol and gain what I want.
But a question :
In trypsin stage my samples that were 30 olds mice clear after 4 days but you told that 1 day is enough for clearing stage.
2/20/2014 3:06:45 AM Reply
Peichuan Zhang
Department of Biology, The Pennsylvania State University, USA

Hi Farzane,

This variation could be due to difference in the efficiency of trypsin digest. Try to adjust the procedures accordingly, as the protocol here serves to provide basic guide line.


2/24/2014 8:09:29 AM

farzane sadeghi
tehran university
a question:
if I want to stain an embryo , I don't need to fix it in neutral bufferd formalin and then washing it with ddH2O and only I need to fix it in ethanol 90% as the first stage.
Is it right according to your protocol?
2/11/2014 4:28:29 AM Reply
farzane sadeghi
tehran university
I want to know that temperature is important in trypsin stage or not.
2/10/2014 11:13:36 AM Reply
Peichuan Zhang
Department of Biology, The Pennsylvania State University, USA

Hi Farzane,

We just soaked the samples in the trypsin solution overnight at room temperature (not at 37 degrees) --- this should work well enough.

For your other question --- we used ethanol instead of formaldehyde to fix embryos. Maybe this helps to avoid over-fixing. I'd recommend you to open the abdomen of the embryos with scissors after sacrificing them in your experiments.


2/13/2014 10:39:36 AM

farzane sadeghi
tehran university
Hello ,
I have a question:
staining the cartilage is done with 0.02% alcian blue (as in your protocol) or 0.2% alcian blue (as in MBC paper)?
2/9/2014 10:26:36 AM Reply
farzane sadeghi
tehran university
I am a basic science in tehran university.
I want to know how long a mice about 30 days old need to be in trypsin solution?
1/31/2014 12:00:09 PM Reply
Peichuan Zhang
Department of Biology, The Pennsylvania State University, USA

Hi Farzane,

We did these experiments a long while ago --- I just checked my thesis and found this note, "samples were further treated overnight with 1.0% trypsin prepared in buffer containing 30% saturated sodium borate", and "when limp and blue cartilage can be readily seen, samples were further treated overnight with Alizarin Red (Sigma, Cat# A-3882" prepared in 0.5% KOH".

So for mice of 30 days old, I'd recommend you to follow the procedures as described in the Bio-Protocol website here. Stain the cartilage with Alcian Blue first, and proceed to the next mineralized bone staining when you can see the cartilage clearly. Prepare a saturated sodium borate solution, and use 30% (volume) to prepare a 1.0% trypsin for digestion overnight. This should allow the Alizarin Red dye penetrate into the bones.


2/5/2014 11:27:49 AM

Can mice of three weeks old be fixed in 95% EOH rather than 10% formalin for this staining procedure?

7/1/2012 1:02:35 PM Reply
Peichuan Zhang
Department of Biology, The Pennsylvania State University, USA


Sorry that I don't really have an answer for you. So we used this protocol to analyze skeletons of both embryos and adults (more than 3-weeks old). We always used 10% buffered formalin for fixation --- I'm not quite sure whether ethanol would serve well. Maybe it can do the job as along as you clean up the tissues as much as possible. Please let us know if you will try ethanol-fixation for adult skeletons, and we will add your input to this protocol then.


7/4/2012 4:13:28 PM

MCB paper is available? can I access?
2/22/2012 2:56:52 PM Reply
Yuanqing Lin

link above

2/22/2012 3:07:10 PM

2/15/2012 9:39:14 AM Reply
Peichuan Zhang
Department of Biology, The Pennsylvania State University, USA

"O/N" is the abbreviation for "overnight". The incubation time is typically ~12 to 16 hours. The staining effects were pretty good, according to my own experience. Please check the MCB paper as reference --- we put a few nice pictures in there.


2/22/2012 9:59:54 AM