[Medicago] Gus staining in Alfalfa

medicago from net.bio.net via medicago%40net.bio.net (by medicago from net.bio.net)
Fri Oct 9 17:15:58 EST 2009


Tim raises a good point regarding pigments.  

In our protocol, we conduct the staining with fresh tissue and then clear 
tissues in 70% ethanol. For keeping stained material for more than a few 
days, place in a fixative/preservative of FAA (50% Ethanol: 85 ml, glacial 
acetic acid: 5 ml, formalin:10 ml).
	
A less noxious preservative is made by mixing 42 mL 95% ethanol, 6 mL 
lactic acid 85% USP, 0.5 g benzoic acid crystals, brought to 100 ml with 
deionized water.

Debby Samac




On Oct 9 2009, medicago from oat.bio.indiana.edu wrote:

>While tissue penetration is part of the problem, the major complication
>is interference by chlorophyll; the effect varies with species and leaf
>age. Etiolation or extraction of chlorophyll with ethanol will allow
>better assessment of promoter activity.
>
>The attached paper "The dark side of green fluorescent protein" from my
>lab (Zhou et al 2005, New Phytol 168:313-322) should be of interest.
>
>Tim 
>
>Timothy C. Hall Ph.D. 
>Distinguished Professor and Director 
>Institute of Developmental and Molecular Biology 
>407 Biological Sciences Building West 
>MS 3155 Texas A&M University 
>College Station, TX 77843-3155 
>
>Phone: 979-845-7728; Fax 979-862-4098 
>tim from idmb.tamu.edu 
>Original Message-----
>From: medicago-bounces from oat.bio.indiana.edu
>[mailto:medicago-bounces from oat.bio.indiana.edu] On Behalf Of
>medicago from oat.bio.indiana.edu
>Sent: Friday, October 09, 2009 12:29 PM
>To: medicago from oat.bio.indiana.edu
>Subject: Re: [Medicago] Gus staining in Alfalfa
>
>Dear Lisa,
>
>My lab has done lots of GUS work with alfalfa, both histochemical and
>enzymatic assays. You are right, the technique you used for Arabidopsis
>does not transfer well to alfalfa. (I could give you quite an extensive
>list of things that don't transfer!) You need to cut the leaves into
>smaller sections, if possible. Whole leaves are almost impossible to
>stain successfully. The key is to do the vacuum infiltration correctly.
>The speed vac is probably pulling a sufficient vacuum, but it is
>releasing the vacuum too slowly. The release needs to be very fast, so
>that the stain permeates into the leaf tissues. I suggest using a small
>side arm flask attached to a vacuum pump. Put a large rubber stopper on
>top of the flask opening but not inside the opening. Pull the vacuum for
>several minutes while swirling the leaf pieces in the stain. You should
>see little air bubbles coming out of the leaf pieces. Then, pull off the
>stopper quickly. You should see the leaf pieces turn darker green as the
>stain infiltrates. You may need to repeat the infiltration process
>several times to get the stain into the center of the leaf piece.
>Incubate at 37 C checking frequently so that over staining does not
>occur. I suggest putting the leaf pieces and stain into a multiwell
>plate that can be put under a dissecting microscope.
>
>Feel free to give me a call to discuss the assay further. I may be able
>to offer other tricks depending on the objectives of your experiment.
>
>Debby Samac
>(612) 625-1243
>
>
>On Oct 8 2009, medicago from oat.bio.indiana.edu wrote:
>
>> We've been trying to do GUS staining in mature Alfalfa plants using a 
>> standard protocol of acetone fix / phosphate buffer &FeCNs & Xgluc & 
>> Tx100 / incubate at 37 / EtOH clear if necessary. This always worked 
>> very well for me in arabidopsis, but in alfalfa, the stain does not 
>> seem to permeate - even in constitutive GUS plants, only the 
>> blade-cut-edges will stain, leading me to believe this is a
>permeability issue.
>>
>> We tried the X-gulc method with cycling vacuum infiltration (which 
>> didn't work either) but I'm not sure what level of low pressure we 
>> achieved (we just used our speedvac). We've also attempted a 
>> fluorometric assay with MUG, which didn't give great results, and may 
>> return to that idea, but visualizing the stain would be much more
>convenient for us.
>>
>> Does anyone have any helpful suggestions on GUS staining permeability 
>> (besides looking at younger plants - this is not an option for this 
>> project)? Thanks! Lisa Koch
>>
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>
>--
>Deborah Samac
>USDA-ARS Research Plant Pathologist
>1991 Upper Buford Circle
>495 Borlaug Hall
>St. Paul, MN 55108
>
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>
>

-- 
Deborah Samac
USDA-ARS Research Plant Pathologist
1991 Upper Buford Circle
495 Borlaug Hall
St. Paul, MN 55108




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