From medicago from net.bio.net Thu Oct 8 16:12:54 2009 From: medicago from net.bio.net (medicago@net.bio.net) Date: Thu Oct 8 17:18:21 2009 Subject: [Medicago] Gus staining in Alfalfa Message-ID: <70c0a82b89c87.4ace0f86@wiscmail.wisc.edu> 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 From medicago from net.bio.net Fri Oct 9 12:29:21 2009 From: medicago from net.bio.net (medicago@net.bio.net) Date: Fri Oct 9 13:11:29 2009 Subject: [Medicago] Gus staining in Alfalfa In-Reply-To: <70c0a82b89c87.4ace0f86@wiscmail.wisc.edu> References: <70c0a82b89c87.4ace0f86@wiscmail.wisc.edu> Message-ID: 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@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 > >_______________________________________________ >Medicago mailing list >Medicago@net.bio.net >http://net.bio.net/biomail/listinfo/medicago > -- Deborah Samac USDA-ARS Research Plant Pathologist 1991 Upper Buford Circle 495 Borlaug Hall St. Paul, MN 55108 From medicago from net.bio.net Fri Oct 9 14:04:19 2009 From: medicago from net.bio.net (medicago@net.bio.net) Date: Fri Oct 9 15:39:43 2009 Subject: [Medicago] Gus staining in Alfalfa Message-ID: <14F3209FBEED5A4DA4569B18B89F564B0AB09E@idmb-exch.idmb.tamu.edu> 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@idmb.tamu.edu Original Message----- From: medicago-bounces@oat.bio.indiana.edu [mailto:medicago-bounces@oat.bio.indiana.edu] On Behalf Of medicago@oat.bio.indiana.edu Sent: Friday, October 09, 2009 12:29 PM To: medicago@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@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 > >_______________________________________________ >Medicago mailing list >Medicago@net.bio.net >http://net.bio.net/biomail/listinfo/medicago > -- Deborah Samac USDA-ARS Research Plant Pathologist 1991 Upper Buford Circle 495 Borlaug Hall St. Paul, MN 55108 _______________________________________________ Medicago mailing list Medicago@net.bio.net http://net.bio.net/biomail/listinfo/medicago From medicago from net.bio.net Fri Oct 9 17:15:58 2009 From: medicago from net.bio.net (medicago@net.bio.net) Date: Sun Oct 11 19:21:42 2009 Subject: [Medicago] Gus staining in Alfalfa In-Reply-To: <14F3209FBEED5A4DA4569B18B89F564B0AB09E@idmb-exch.idmb.tamu.edu> References: <14F3209FBEED5A4DA4569B18B89F564B0AB09E@idmb-exch.idmb.tamu.edu> Message-ID: 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@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@idmb.tamu.edu >Original Message----- >From: medicago-bounces@oat.bio.indiana.edu >[mailto:medicago-bounces@oat.bio.indiana.edu] On Behalf Of >medicago@oat.bio.indiana.edu >Sent: Friday, October 09, 2009 12:29 PM >To: medicago@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@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 >> >>_______________________________________________ >>Medicago mailing list >>Medicago@net.bio.net >>http://net.bio.net/biomail/listinfo/medicago >> > >-- >Deborah Samac >USDA-ARS Research Plant Pathologist >1991 Upper Buford Circle >495 Borlaug Hall >St. Paul, MN 55108 > >_______________________________________________ >Medicago mailing list >Medicago@net.bio.net >http://net.bio.net/biomail/listinfo/medicago > > > -- Deborah Samac USDA-ARS Research Plant Pathologist 1991 Upper Buford Circle 495 Borlaug Hall St. Paul, MN 55108