Has anyone a good tip to do nice mounted sample for immunocytochemistry? I have tried to have good images, but, sometimes, my embryos are close to the boundary of the mounting reagent and I have problems with fluorescence there; other times I have bubbles, even when I try not to form them when I put the embryo over the slipe or put the coverslip. Sometimes the mounting reagent deform the shape of my embryos and they dont look well!, I need help.
One other question: is there a way to replace the mounting reagent (vectashield or the other of invitrogen that are both very expensive) for some lab preparation that conserves fluorescence for several months?
I' have had problems with gilcerol too! don`t you? don`t they change their shape also in glicerol?
HI,
I´m also working with embryos (morulae) and fluoresecence. At beginig I started mounting my embryos with glycerol using 1:1 dilution , but currently I´m going to change to DAKO. May be it would be helpful I sent you the website so that you could check it.
http://www.dako.com/dist/ar38/p107570/prod_products.htm
Regards,
I don't mount anything large like embyos. Maybe you need to use lab tape and build up a boundary on the edge of your slide so that the embryos will be in an equal volume and not be deformed by the cover slip. I use Mowiol. A bit time consuming to make, 48 hours, but you can make enough to give you a 6 months supply, just freeze the extra at -80C.
Mowiol Mounting Medium
1g Mowiol (solid) dissolved in 4 ml phosphate buffer (0.015M NaPO4 pH 8.0)
Stir overnight at room temperature. Cover with parafilm so buffer doesn't evaporate. Solution will be very thick. Stir slowly so not to create bubbles.
Slowly add 2ml 100% glycerol. Stir overnight at room temperature. Centrifuge 12,000rpm (in 35ml tubes, Sorval SS34 rotor) 15min, 4C. Pour off supernatent. Will be a pellet of undissolved Mowiol. Mark the outside of the tubes as the pellet is difficult to see. Aliquot in 5ml portions for long term storage at -70C. Keep the tube being used at -20C, ie short term storage.
I typically scale up this protocol and make about 50 ml at a time. Each coverslip needs only one drop from a pipette tip, probably only 50ul.
Hope this helps. Fluorescence is stable for more than 1 year.
HAS ANYBODY ACCES TO THIS ARTICLE THAT CAN PROVIDE ME!?
http://cshprotocols.cshlp.org/content/2010/5/pdb.prot5426.full.pdf+html
my mail: [email protected]
thanks!
Dear Jesica,
I would like to propose you should write an e-mail to the corresponding author (see below). This might be the fastest way to get the article (if nobody responds earlier to your request). I do have good experiences in writing to corresponding authors, just telling them a little bit about WHO you are (student...etc.) and the goal of your recent and present work...Unfortunately the article also for me only accessible via prepaid view (US 20.- + taxes).
I have copied the Ttitle as well as the affiliation and e-mail address of the corresponding author for you:
Protocol
Preparation of Fixed Xenopus Embryos for Confocal Imaging
Abstract: http://cshprotocols.cshlp.org/content/2010/5/pdb.prot5426.abstract
John B. Wallingford
+ Author Affiliations: Howard Hughes Medical Institute and Section of Molecular Cell and Developmental Biology, University of Texas, Austin, TX 78712, USA
Corresponding author ([email protected]).
Best wishes and regards,
Wolfgang MUSS PhD
SALZBURG, AUSTRIA
What Wolfgang said is good advice in general... I email people all the time asking for papers and 99% of the time they are happy to forward me the PDF.
yeah! I usually do that too! but this time they havent answered me yet! :S
Hi, I wonder if cavity, or concavity slides like the ones in this link would help protect your samples?
https://uk.vwr.com/app/catalog/Product?article_number=631-0482
For large samples I wouldn't recommend a glycerol based agent unless you have a glycerol corrected objective, and even then not great for thick specimens. I would clear in a benzyl alcohol benzyl benzoate solution (BABB) or methyl salicylate following dehydration in an ethanol series. These clear large specimens very well and have a refractive index much closer to oil immersion objectives. If the sample if getting distorted then cavity slides as recommended above can be used or you can place coverslips either side of your sample to prevent the top coverslip pushing on the specimen - be careful not to make the distance too great though so that you can't focus on it anymore due to exceeding the working distance of the objectives on your microscope.
For long term storage I would clear in BABB and then mount in DPX mounting solution. We store alexafluor stained embryos this way and find they are still usable months later.
Mowiol-with antifade (DABCO or NPG)
2.4 g of Mowiol-88 (sigma),
6 g of glycerol
6 mL of H2O.
Mix for 3 hours at room temp
Add 12 mL of 0.2 M Tris-Cl (pH 8.5). Incubate with mixing at 50°C for 10 min. (I've also mixed over night on a blood roller in a 50ml falcon).
Centrifuge at 5000g for 15 min to pellet insoluble material. (there will be quite a lot).
Add antifade to final concentration of 2.5% for DABCO or 2% for npg. Store in 500-μL aliquots at −20°C.
thaw to use, store at 4C or -20C
Add 5ul per coverslip ( I use 15mm circular) onto glass slide, invert coverslip onto drop
Remove extra mowiol carefully around edges with a little aspiration or by capillary action with a kim wipe.
place in drawer overnight to harden
(if you're in a hurry.... place at 37C for an hour to harden)
store slides at 4C in slide box.
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