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We were always taught when preparing specimens for EM preparation to avoid long periods in low alcohol concentrations - 50% and 70% - as these would strip lipids esp. membranes and degrade the ultrastructure. 

Perhaps the same is happening here, you could try either keeping the specimens in formalin or in higher ethanol concentrations. Another alternative could be PBS-azide with a low BSA and storage in the fridge.

Cheers
 
Ray G


-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Fulvio Florenzano
Sent: Tuesday, 26 August 2008 7:38 p.m.
To: [log in to unmask]
Subject: R: DAPI Staining Issue

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Hi Farid,

Your description sounds like extraction or dislocation of cellular
components by prolonged exposure to alcohol or by using wrong alcohol
concentrations. Alcohol is a solvent for lipids and is able to affect the
morphology of lipidic cellular components, typically membranes. The poor
nuclear morphology evidenced with two different nuclear stains is a strong
indication that your material is morphologically compromised. Try to work
only on your best material and discard the compromised material. 

Regards

Fulvio
 
Fulvio Florenzano PhD
Assistant Imaging Unit 
Lab. of Experimental Neurorehabilitation
"S. Lucia" Foundation
Via fosso del Fiorano, 64/65
00143 Rome Italy
e-mail: [log in to unmask]
lab phone: +39-6-501703080
Fax: +39-6-501703305
mobile: 333-2125293

 -----Messaggio originale-----
Da: Confocal Microscopy List [mailto:[log in to unmask]] Per
conto di Farid Jalali
Inviato: martedì 26 agosto 2008 3.42
A: [log in to unmask]
Oggetto: Fwd: DAPI Staining Issue


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Hello Group,


Not a confocal microscopy question but a sample preparation question; I have
a colleague who is getting very poor nuclear morphology from tumour
xenografts that are fixed in 10% neutral buffered formalin for 24 hours, and
then transferred to 70% ethanol. They are subsequently paraffin embedded and
sectioned to 4um. The H and E stains also reflect poor nuclear morphology
but it is most clearly evident when the sections are stained with 0.1ug/ml
DAPI and mounted with Vectashield for widefield or confocal imaging. We are
accustomed to seeing nuclei that are generally homogeneously stained, with a
couple of nucleoli that are well defined. The problematic sections seem more
vacuolar in appearance. These tumours do not have as many nucleoli as the
image may suggest. Also, when stained with a marker that forms punctate foci
within nuclei, the marker seems to accumulate on the edges of the more dense
DAPI staining, rather than be distributed through the nucleus.

Any thoughts on this sample prep question would be greatly appreciated. I
know there are many of you in this forum with expertise in tissue
preparation. I have 2 images, one of good DAPI/nuclear morphology and one of
poor; what is the convenient way let the group have a look at what I am
trying to explain? Thanks very much to all. 

Cheers
Farid

-- 
Farid Jalali MSc
Senior Research Technician- Lab Manager
Applied Molecular Oncology/ Princess Margaret Hospital
STTARR Innovation Facility/ Radiation Medicine Program
Toronto, Canada
416-946-4501 X4351 (Princess Margaret Hospital)
416-581-7754 STTARR at MaRS Building
416-581-7791 STTARR Microscopy Suite