gus staining chloroplast wash off

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gus staining chloroplast wash off

Chou Hsuan

Hi all,
I have a problem with washing off chloroplasts after gus staining.I tried different protocols- different  concentration (50%, 70%) ethanol to wash off chloroplasts, but it seems that the leaves still look a little greenish (or yellowish) after that.Is there a good way of washing off the chloroplasts and get good pictures like those published in journals?Thank you very much for answering my questions.
Tina
     
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Re: gus staining chloroplast wash off

Rosemary White
Hi Tina,

After the 70% ethanol wash following GUS staining, you need to go to 95%
for at least 1 h - we often leave overnight, then 100% ethanol, may need
to change the 100% wash a couple of times. You'll see the pigments come
out at these higher solvent levels.

Then for long-term storage, if you need it, and for image capture, go back
to 70% ethanol - the tissue is too brittle at the higher solvent levels.


In our hands, it's more effective to go to 95% then 100% rather than
straight from 70% to 100%. Acetone is a better extracting solvent for
these pigments but it's considered quite toxic these days so try to stick
with ethanol.

cheers,
Rosemary

Dr Rosemary White
CSIRO Plant Industry
GPO Box 1600
Canberra, ACT 2601
Australia

T 61 2 6246 5475
F 61 2 6246 5334
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On 15/05/12 4:03 AM, "Chou Hsuan" <[hidden email]> wrote:

>
>Hi all,
>I have a problem with washing off chloroplasts after gus staining.I tried
>different protocols- different  concentration (50%, 70%) ethanol to wash
>off chloroplasts, but it seems that the leaves still look a little
>greenish (or yellowish) after that.Is there a good way of washing off the
>chloroplasts and get good pictures like those published in journals?Thank
>you very much for answering my questions.
>Tina
>    
>_______________________________________________
>Arab-gen mailing list
>[hidden email]
>http://www.bio.net/biomail/listinfo/arab-gen


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