DABCO-mounting medium
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Hi everyone,
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Louis, This is from my notes, info from the Confocal listserver ~10yr ago, and some notes of my own. Hope this helps, Dale ---------------------------- Anti-Fade Formulations <><><><><><><><><><><> Here are a couple of formulas for anti-(photobleach)fade reagents. I got these off the Confocal Listserver a few years back. The people on the list judged them favorably compared with commercial reagents like VectaShield and Molecular Probes "Slo-Fade" anti-fades, but there may be exceptions. Molecular Probes now has several formulations on the market. But Molecular Probes also has some info showing that even their own anti-fades diminished the photostability (over PBS) when used with the AlexaFluor dyes. Some anti fades from MP give a slower decay, but at the expense of lower initial fluorescence. ------------------------------------------------------- Anti-fade reagents to reduce photobleaching rates. These reagents are available from Sigma, and others. 1% DABCO in 90% glycerol10% PBS (neutral pH). 4% n-propyl gallate in 90% glycerol10% PBS (neutral pH) (1-2 hr to dissolve; filter through coarse paper; store in brown bottle at RT) (Giloh and Sedat; Science, 1982) Louis Villeneuve wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hi everyone, > > I am looking for a recipe for mounting media with DABCO diluted in > glycerol. The one I have is DABCO, 0.4% (diluted in PBS) mix 1:4 in > glycerol. Also, where I could find a database for finding the > refractive index of several mouting medium/media? > > Thanks > > Louis Villeneuve > > Louis Villeneuve > Research Associate - Confocal Microscopy > Heart Montreal Institute- Research Center > 5000 East Belanger > Montreal (Qc), Canada > H1T 1C8 > > 514-376-3330 ext 3511 > 514-376-1355 (Fax) > > [hidden email] > |
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In reply to this post by Louis Villeneuve
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi I use DABO in a formulation of 1% DABCO in 85 - 90 % glycerol, with 10 - 15 % PBS if I use FITC I adjust the pH to 8.0 - 8.5 using indicator strips, as FITC is noticably brighter at a higher pH. Russ Russell N. Spear Sr. Research Specialist Dept. of Plant Pathology Univ. of Wisconsin 1630 Linden Dr. Madison WI 53706 voice 608.263.2093 fax 608.263.2626 >>> Louis Villeneuve <[hidden email]> 03/11/08 8:14 AM >>> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi everyone, I am looking for a recipe for mounting media with DABCO diluted in glycerol. The one I have is DABCO, 0.4% (diluted in PBS) mix 1:4 in glycerol. Also, where I could find a database for finding the refractive index of several mouting medium/media? Thanks Louis Villeneuve Louis Villeneuve Research Associate - Confocal Microscopy Heart Montreal Institute- Research Center 5000 East Belanger Montreal (Qc), Canada H1T 1C8 514-376-3330 ext 3511 514-376-1355 (Fax) [hidden email] |
![Indig, Fred (NIH/NIA/IRP) [E]](/images/avatar100.png "Indig, Fred (NIH/NIA/IRP) [E]"){kind=avatar} 
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Indig, Fred (NIH/NIA/IRP) [E] |
Re: DABCO-mounting medium
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In reply to this post by Louis Villeneuve
Search the CONFOCAL archive at
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Dear Louis,
I have used the following formula for over 10 years (Indig et
al., Biochem J. 327:291-8, 1997) with excellent results:
(1) Dissolve in beaker (0.5 L for 100
mL solution):
(2) This could take a while (hours)- use a heated magnetic
stirrer at low speed, about 50 C.
(3) After all is dissolved, aliquot 0.25 mL into microtubes,
store -20 C in the dark (good for well over one year).
(4) Before use, heat to 65 C for 10 min, let cool 1-2 min and use
immediately on specimen. Discard remains. This mounting media will
solidify overnight in the refrigerator (4 C, dark), but is not as hard
as epoxy mounts, so handle gently.
(5) I measured the refractive index as R=1.4101 @ 22.0 C (water
1.33, oil 1.56)
Best Wishes,
Fred
Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi everyone, -- Fred E. Indig, Ph.D.
Head, Confocal Imaging Facility Research Resources Branch
NIH Biomedical Research
Center (BRC)
National Institute on
Aging,
251 Bayview
Boulevard,
Room #4B026
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In reply to this post by Louis Villeneuve
Search the CONFOCAL archive at
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Hi Louis,
Here's the recipe we recommend to users, adapted from: Johnson, GD, Davidson, RS, McNamee, KC, Russell, G, Goodwin, D, and Holborow, EJ, Fading of Immunofluorescence during microscopy: a Study of the Phenomenon and its Remedy, Journal of Immunological Methods, 55 (1982)231-242. Materials: 1,4-Diazobicyclo-(2,2,2)octane (Sigma #D2522) Glycerol, spectorophotometric grade (Kodak) 1XPBS HCl (diluted 1:20 with H20) Methods: Mix 25 mg/ml of DABCO in 90% glycerol/10% 1xPBS. You may gently heat and or rock the mixture to dissolve. Adjust pH to 8.6 with diluted HCl. For example, to make 25 ml final volume: 625 mg Sigma DABCO 22.5 ml glycerol 2.5 ml 1xPBS Adjust pH to pH 8.6 with diluted HCl. Dave At 07:14 AM 3/11/2008, you wrote: Hi everyone, Scientific Imaging Lab Fred Hutchinson Cancer Research Center 1100 Fairview Avenue North, DE-512 Seattle, WA 98109 206-667-4205 http://www.fhcrc.org |
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In reply to this post by Louis Villeneuve
Search the CONFOCAL archive at
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Hi Louis,
list of R.I.'s of variety of things: http://en.wikipedia.org/wiki/List_of_refractive_indices data for RI's of mounting media are in the refractive index section of the word doc inside http://home.earthlink.net/~mpmicro/mpmicro.zip Enjoy, George At 10:14 AM 3/11/2008, you wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal George McNamara, Ph.D. University of Miami, Miller School of Medicine Image Core Miami, FL 33010 [hidden email] [hidden email] 305-243-8436 office http://home.earthlink.net/~pubspectra/ http://home.earthlink.net/~geomcnamara/ http://www.sylvester.org/research/SR_lab_analytical.asp?ana=desc (Analytical Imaging Core Facility) |
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cheers, rosemary On 7/4/08 7:32 AM, "George McNamara" <[hidden email]> wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Louis, |
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