Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Farid,
I agree with each of the responses to your question, which to summarize:
1) use paraformaldehyde made fresh which provides better more
extended crosslinking because it remains a polymer for awhile until
reverting to formaldehyde monomers.
2) proper dehydration is essential if using EtOH. This means ~70%
EtOH, 80%, 90%, and finally absolute EtOH. This can still cause
serious artifacts because of rearrangement of lipid membranes and
extraction of some proteins and lipids. Without complete dehydration,
the paraffin embedding can create apparent vacuolar structures.
3) Polylysine (PL) can promote dye sticking if the polylysine has not
been fully blocked. The PF-amino binding is unstable unless the
Schiff's base is reduced say with sodium borohydride or similar
reducing agent. The polyamino groups can lead to electrostatic
binding of negatively charged stains. This can be prevented by
treating with fresh dilute (~ 1 mM) succinic anhydride or acetic
anhydride before staining.
4) I have always had severe background staining using plastic
coverslips with or without PL. Good for growing cells but their
hydrophobicity really causes problems with a lot of stains because of
the amphoteric nature of most dye molecules. I gave up on plastic
decades ago. PL and 5 ug/ml fibronectin on coverslips attach cells
for growing is good most the cell types I've used.
5) The H & E staining even if you are not doing the unfortunate
paraffin embedding, will never give good nuclear DNA staining
compared to DAPI, Hoechst, and Draq5. The two former stains are minor
groove labels that work very well for nuclear DNA staining. Almost
all other nucleic acid stains label all nucleic acids whether they
are mitochondrial DNA, RNA, or any other _NA. I also agree that using
a much higher DAPI concentration is sensible. Given the density of
DNA in the nucleus you have to worry about saturating the DNA. Flow
Cy. commonly uses 3 ug/ml which makes for reasonably quantitative
analysis and growth stage evaluation using flow.
I have to stop no, but feel free to email me the images you are
having problems with. I am curious to see them.
good luck,
Mario
[log in to unmask]
[log in to unmask]
[log in to unmask]
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>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
|
