antibodies to DsRed
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Dear list,
A colleague is experiencing severe losses of fluorescence in DsRed after PFA fixation. He is working with Drosophila imaginal discs and would like to know about other methods of fixation or of good specific antibodies to DsRed. Anyone can help? Thanks, -- F Javier Díez Guerra, PhD Profesor Titular Centro de Biología Molecular Severo Ochoa C/ Nicolás Cabrera, 1 Universidad Autónoma Ctra Colmenar Viejo Km 15 Cantoblanco, 28049 Madrid SPAIN phone: +34 91 196 4612 e-mail: [hidden email] |
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Rockland polyclonal to RFP works with PFA fixation. Recognizes dsRed,
mRFP1, tdTomato, mApple-probably others. We use it routinely. No commercial interest. Mike Michael J. Schell, Ph.D., CIV, USUHS Assist. Professor Dept. of Pharmacology Uniformed Services University 4301 Jones Bridge Rd. Bethesda, MD 20814-3220 tel: (301) 295-3249 [hidden email] >>> F Javier Díez Guerra <[hidden email]> 04/21/10 9:43 AM >>> Dear list, A colleague is experiencing severe losses of fluorescence in DsRed after PFA fixation. He is working with Drosophila imaginal discs and would like to know about other methods of fixation or of good specific antibodies to DsRed. Anyone can help? Thanks, -- F Javier Díez Guerra, PhD Profesor Titular Centro de Biología Molecular Severo Ochoa C/ Nicolás Cabrera, 1 Universidad Autónoma Ctra Colmenar Viejo Km 15 Cantoblanco, 28049 Madrid SPAIN phone: +34 91 196 4612 e-mail: [hidden email] Classification: UNCLASSIFIED Caveats: None |
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Thanks, Michael.
Javier El 21/04/2010 15:56, Michael Schell escribió: > Rockland polyclonal to RFP works with PFA fixation. Recognizes dsRed, > mRFP1, tdTomato, mApple-probably others. We use it routinely. No > commercial interest. > Mike > > Michael J. Schell, Ph.D., CIV, USUHS > Assist. Professor > Dept. of Pharmacology > Uniformed Services University > 4301 Jones Bridge Rd. > Bethesda, MD 20814-3220 > tel: (301) 295-3249 > [hidden email] > >>>> F Javier Díez Guerra<[hidden email]> 04/21/10 9:43 AM>>> >>>> > Dear list, > > A colleague is experiencing severe losses of fluorescence in DsRed after > > PFA fixation. He is working with Drosophila imaginal discs and would > like to know about other methods of fixation or of good specific > antibodies to DsRed. > > Anyone can help? > > Thanks, > |
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In reply to this post by F Javier Díez Guerra
Presence of solvents (alcohol, acetone, organic acids, nail polish) will severely affect many GFPs. GFPs are also sensitive to pH. Therefore i would recommend checking for those. We did fix EGFP in imaginal discs with 3.5% PFA at pH 7.4-8 with good results. DsRed could be more sensitive to fixation than EGFP (I have no experience with DsRed fixation), but the reason for the loss of fluorescence could be more trivial, so better exclude those first...
-- Julio Vazquez Fred Hutchinson Cancer Research Center 1100 Fairview Ave. N., mailstop DE-512 Seattle, WA 98109-1024 On Apr 21, 2010, at 6:17 AM, F Javier Díez Guerra wrote:
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Another thing to check is the age of the PFA. Old PFA will break
down and quench fluorescence very well. Best to use PFA that comes in ampoules stored
under nitrogen. That way you know when you rack one open it is totally fresh.
Once opened you can store is for a week or so in a plastic tube at 4 degrees
wrapped in foil. Cheers Cam Cameron J. Nowell Office: +61 3 9341 3155 From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Julio Vazquez Presence of solvents (alcohol, acetone, organic acids, nail
polish) will severely affect many GFPs. GFPs are also sensitive to pH.
Therefore i would recommend
checking for those. We did fix EGFP in imaginal discs with 3.5% PFA at pH 7.4-8 with good results. DsRed
could be more sensitive to fixation than EGFP (I have no experience with DsRed
fixation), but the reason for the loss of
fluorescence could be more trivial, so better exclude those first... -- Julio Vazquez Fred Hutchinson Cancer Research Center 1100 Fairview Ave. N., mailstop DE-512 Seattle, WA 98109-1024
On Apr 21, 2010, at 6:17 AM, F Javier Díez Guerra wrote:
Dear list, A colleague is experiencing severe losses
of fluorescence in DsRed after PFA fixation. He is working with Drosophila
imaginal discs and would like to know about other methods of fixation or of
good specific antibodies to DsRed. Anyone can help? Thanks, This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research Ltd does not waive any rights if you have received this communication in error. The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research Ltd. |
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Can you give me a vendor/product number? I’d like to test some out if I can. Caroline On 4/21/10 6:56 PM, "Cameron Nowell" <Cameron.Nowell@...> wrote: Another thing to check is the age of the PFA. Old PFA will break down and quench fluorescence very well. Best to use PFA that comes in ampoules stored under nitrogen. That way you know when you rack one open it is totally fresh. Once opened you can store is for a week or so in a plastic tube at 4 degrees wrapped in foil. |
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Hi Caroline, Here you go http://www.emsdiasum.com/microscopy/products/chemicals/formaldehyde.aspx#15700 The one you want is the 10x10ml (cat# 15710) Cheers Cam From: Confocal Microscopy List
[mailto:[hidden email]] On Behalf Of Caroline Bass Hi Cam, Another thing to check is the
age of the PFA. Old PFA will break down and quench fluorescence very well. Best
to use PFA that comes in ampoules stored under nitrogen. That way you know when
you rack one open it is totally fresh. Once opened you can store is for a week
or so in a plastic tube at 4 degrees wrapped in foil. This communication is intended
only for the named recipient and may contain information that is confidential,
legally privileged or subject to copyright; the Ludwig Institute for Cancer
Research Ltd does not waive any rights if you have received this communication
in error. This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research Ltd does not waive any rights if you have received this communication in error. The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research Ltd. |
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