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Alcian Blue – Alizarin Red Staining of Mouse Skeleton   

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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.

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
Hello
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
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
2/20/2014 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.

Thanks,
P

2/24/2014 8:09:29 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?
2/11/2014 4:28:29 AM Reply
farzane sadeghi
tehran university
Hello
I want to know that temperature is important in trypsin stage or not.
Thanks
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.

Best,
P

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)?
Thanks
2/9/2014 10:26:36 AM Reply
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?
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.

Best,
Peichuan

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?

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

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