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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.

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Alcian Blue – Alizarin Red Staining of Mouse Skeleton
阿尔辛蓝 - 茜素红对小鼠骨架染色

发育生物学 > 形态建成
作者: Peichuan Zhang
Peichuan ZhangAffiliation 1: Department of Biology, The Pennsylvania State University, University Park, PA, USA
Affiliation 2: Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA
For correspondence: peichuan.zhang@ucsf.edu
Bio-protocol author page: a11
Vol 2, Iss 8, 4/20/2012, 15368 views, 9 Q&A
DOI: https://doi.org/10.21769/BioProtoc.162

[Abstract] 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.

[Abstract] 我们的实验室已使用经一些改进的阿尔辛蓝 - 茜素红染色法[1]来描绘因缺乏Pek/Perk的小鼠骨骼畸形。这个基因编码了调控翻译的eIF2α激酶 [2]。

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)

Equipment

  1. Conical tube

Procedure

  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.

Recipes

  1. Trypsin solution
    1.0% trypsin
    30% saturated sodium borate
    H2O

Acknowledgments

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.

References

  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.

材料与试剂

 

1.         神经福尔马林 (Sigma, catalog# HT50-1-4)

2.         阿尔辛蓝 8GX (Sigma, catalog# A-5268)

3.         胰蛋白酶 (Sigma, catalog# T1426)

4.         饱和四硼酸钠(Sigma, catalog# S-9640)

5.         茜素红 (Sigma, catalog# A-3882)

6.         麝香草酚 (EM Science, catalog# TX0615-1)

7.         乙醇

8.         冰醋酸

9.         氢氧化钾

10.      甘油

11.      ddH2O

 

器材:

 

1.         锥形管

 

步骤:

 

1.          成年鼠:

1)       将小鼠骨架固定在10%中性福尔马林缓冲液中至少24小时。

2)     ddH2O O / N1小时胚胎)清洗样品,轻轻摇动,并在之后存于70%的乙醇

注:此时,样品可以在70%乙醇保存期长。

3)       小心从样品中取出皮肤和内脏。
注:清除所有的皮肤,甚至那些小脚趾上的。

4)       0.02阿尔新蓝8GX(乙醇/冰醋酸,7:3配制染色样品12天。
注:软骨组织被染成蓝色。  

5)       用纯乙醇/冰醋酸(7:3洗样品1小时。

6)       将样品浸泡在100%的乙醇O / N,然后ddH2O12天。

7)       把样品用1.0%胰蛋白酶O / N30%的饱和硼酸钠水溶液)处理样品

8)       此时,和蓝色软骨很容易观察,接下来用茜素红O / N(用0.5KOH配制染样品。
注:补充足够的(没有具体数值)饱和茜素红直到溶液变为暗紫色。矿化骨骼将被染成红色。

9)       0.5KOH/甘油的梯度系列(即2:11:11:2100%的甘油,每一步2天)处理样品,并存储在含晶体的甘油麝香草酚。

2.          胚胎:

1)         类似的方法可用于染色胚胎。为了这一步实验需要将胚胎置于90%的乙醇至少1周。

2)         0.01%阿尔辛蓝8GX处理样品3天,然后通过乙醇梯度系列溶液使样品再水化。70%乙醇,2-3hrs小时,240%乙醇,2-3hrs15%的乙醇, 2-3hrs ddH2O直到样品沉淀到锥形管底部)。

3)         新鲜的1KOH处理样品1-2,直到它变得清

4)         0.001%茜素红处理样品2-3天,直到骨变成紫色

5)         1KOH清洗样品3,每次几个小时。

6)         甘油-KOH度系列处理样品 20甘油/1KOH24hrs50%甘油/ 1KOH24hrs80%甘油/1KOH24hrs100%甘油24hrsx2)。Treat the sample through a gradient series of glycerol - KOH (20 % glycerol/ 1 % KOH, 24 hours; 50 % glycerol/ 1 % KOH, 24 hours; 80 % glycerol/ 1 % KOH, 24 hours; 100 % glycerol, 24 hours X 2). 

 

配方:

 

1.         胰蛋白酶溶液

1.0 %胰蛋白酶

30% 饱和四硼酸钠

H2O

 

参考文献:

 

1.        Hanken J., Wassersug R.J. (1981). The visible skeleton. Functional photography 16(4): 22-26,44.

2.        Zhang P., McGrath B., Li S., Frank A., Zambito F., Reinert J., Gannon M., Ma K., McNaughton K., 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. Molecular and Cellular Biology 22(11): 3864-74. 

 

 

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How to cite this protocol: Zhang, P. (2012). Alcian Blue – Alizarin Red Staining of Mouse Skeleton. Bio-protocol 2(8): e162. DOI: 10.21769/BioProtoc.162; Full Text



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3/3/2014 8:26:32 AM  

farzane sadeghi
tehran university

Hello
can we remove skin and internal organ at first and then fix the samples?

Reply

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2/20/2014 3:06:45 AM  

farzane sadeghi
tehran university

Hello
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.
why?
Thanks

2/24/2014 8:09:29 AM  

Peichuan Zhang (Author)
Department of Biology, The Pennsylvania State University, University Park, 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.

Thanks,
P

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2/11/2014 4:28:29 AM  

farzane sadeghi
tehran university

Hello
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?

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2/10/2014 11:13:36 AM  

farzane sadeghi
tehran university

Hello
I want to know that temperature is important in trypsin stage or not.
Thanks

2/13/2014 10:39:36 AM  

Peichuan Zhang (Author)
Department of Biology, The Pennsylvania State University, University Park, 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.

Best,
P

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2/9/2014 10:26:36 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)?
Thanks

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1/31/2014 12:00:09 PM  

farzane sadeghi
tehran university

Hello
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?

2/5/2014 11:27:49 AM  

Peichuan Zhang (Author)
Department of Biology, The Pennsylvania State University, University Park, 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.

Best,
Peichuan

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7/1/2012 1:02:35 PM  

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

Thanks.

7/4/2012 4:13:28 PM  

Peichuan Zhang (Author)
Department of Biology, The Pennsylvania State University, University Park, USA

Hello,

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.

Thanks,
Peichuan

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2/22/2012 2:56:52 PM  

MCB paper is available? can I access?

2/22/2012 3:07:10 PM  

Anonymous Lin
Test institute

link above

Reply

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2/15/2012 9:39:14 AM  

O / N是生命意思啊?
实验效果怎么样啊?

2/22/2012 9:59:54 AM  

Peichuan Zhang (Author)
Department of Biology, The Pennsylvania State University, University Park, 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.

Thanks,
Peichuan

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