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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi Caroline,
Your images at
http://homepage.mac.com/etlab/photos-liver/PhotoAlbum4.html look good
(publishable ... well, no need to publish the GFP acetone fixed, or
RFP/nbf fixed, not too excited by the RPF/nbf+acetone).
I'd still with frozen specimens or fresh tissue, if possible. The
RFP/NBF fixed might be recovered by immunofluorescence with an
anti-DsRed antibody (Cy3 or if you can find it on hand, Cy3B
secondary antibody, or to change color a bit, Alexa Fluor 647
secondary antibody). the people I work(ed) with at CHLA routinely
detected EGFP on fixed specimens by immunofluorescence.
Your observation that your direct imaging (on your lab widefield
scope?) had RFP much better than the GFP, and opposite results on the
confocal, is probably due to:
* having an ideal RFP filter set on your scope, and relatively
mediocre filter set for GFP -- perhaps you are using an old
fluorescein set ... might be time to check if the filters are still
good (exciter gets solarized over time, fingerprints can magically
appear on any of the surfaces).
* the confocal is working well with EGFP because it has a 488 nm
laser - I'm guessing you have a low power HeNe 543 nm laser for the
RFP or are trying to excite RFP with 488 nm? ... if you are using
simultaneous scanning for both, i.e. through a triple 488/543/633
filter, I suggest sequential scanning with optimal dichroics. If a
confocal facility person is running the instrument, I suggest sitting
with then and finding the optimal setting for RFP by using a specimen
that you've seen worked well on your scope.
Best wishes,
George
At 11:19 AM 11/20/2005, you wrote:
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Hello George,
>
>Thanks for the advice. I have already processed some tissue, and I
>know I have expression, but now I have to make some comparisons. I
>have given some tissue, both frozen liver and small blocks fixed by
>immersion in NBF to our confocal core facility. I was concerned
>about the fixation, or lack thereof, for visualizing native
>fluorescence. I just got the photos back and I'm not sure what to
>make of them. For some reason the guy acetone fixed some of the
>section, which I don't understand because I thought acetone killed
>EGFP. The results show bad EGFP fluorescence with acetone fixation,
>but the best RFP signal was with acetone. He also let the section
>dry on the slide. So now I am totally confused.
>
>Perhaps you could take a look. I just posted the pics on the web.
>Click on the thumbnails for better photos.
>
>http://homepage.mac.com/etlab/photos-liver/PhotoAlbum4.html
>
>I don't want to bug you with this. I appreciate your advice,
>especially on a Sunday. I will probably try DAPI, as I believe I
>already have some in the lab. I have also checked out the dishes you
>recommended. They give out samples so it will be very helpful I
>think. I actually tried the razor blade methods, which I called
>liver squishes. I just added a little bit of water and squished the
>coverslip down. The real problem I have is that the RFP was
>terrific, much better than the EGFP. But I get the total opposite
>from the confocal images. Initially I thought that perhaps I cut a
>thicker chunk off the RFP liver resulting in what looked like more
>positive cells, but now I wonder. I have to be able to reliably
>figure out the percentage of positive cells, so the razor blade
>method may be a problem. However, if the DAPI works I suppose I
>could just count positive cells/nuclei.
>
>Thanks for your help!
>
>Caroline
>
>
>
>
>
>On Nov 20, 2005, at 1:59 PM, Mcnamara, George wrote:
>
>>Search the CONFOCAL archive at
>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>>Hi Caroline,
>>
>>Good EGFP expression will result in brilliant neon green
>>fluorescence, as
>>opposed to the dimmer yellow green of control tissue. Best to place
>>both
>>your vector labeled and a negative control tissue section on the
>>same slide
>>(use a big coverslip). Be sure to keep the tissue sections wet -
>>dehydration
>>kills GFP. Your best bet with tissue is to use a cryostat to cut
>>fresh (not
>>frozen) 5 or 10 um sections (a vibratome can be used instead). In
>>fact, even
>>simpler (on an inverted scope) is to slice the liver with a razor
>>blade, put
>>it on a microscope slide or better yet imaging dish (i.e. mattek
>>dishes at
>>http://www.glassbottomdishes.com <http:// www.glassbottomdishes.com> ) cover
>>it with saline to keep it moist, put it on the microscope and look.
>>You can
>>see EGFP+ cells to about 20 um deep with a widefield fluorescence
>>scope, ~50
>>um deep with confocal (excitation 488 nm).
>>
>>DAPI or Hoechst 33342 or 33258 are both good choices (assuming UV
>>excitation/blue or 400LP emission filter set). A few minutes in
>>Hoechst at 1
>>to 10 ug/mL is sufficient to label all the nuclei (give razor
>>slices 30
>>minutes). Then do a quick rinse of the slide with saline or
>>transfer the
>>slice to an imaging dish with saline. If you start with a 10 mg/mL
>>stock
>>solution of Hoechst (Invitrogen/Molecular Probes) be sure to dilute
>>100 fold
>>into dH20 (Hoechst into PBS makes pretty precipitate, but does not
>>help in
>>seeing cells).
>>
>>H&E would be a mistake - the standard dehydration kills EGFP (which
>>can then
>>be imaged by immunofluorescence using anti-GFP and Alexa Fluor 488
>>secondary
>>antibody, but this adds more variables to what should be simple),
>>hematoxylin would absorb some of the DAPI or Hoechst signal, and
>>eosin has
>>bright yellow (dim with a narrow pass EGFP filter set, but could be a
>>problem with a wider emission filter set).
>>
>>To help distinguish EGFP from autofluorescence I like to image the
>>latter
>>with a Chroma ECFP filter set (Chroma 31044v2: D436/20x, 455DCLP,
>>D480/40m).
>>Anything that fluoresces in both the EGFP and ECFP sets should be
>>suspected
>>as autofluorescence. This assumes you have a set. On a confocal, a
>>405, 440,
>>or 458 nm laser line can be used for autofluorescence, with
>>emission set to
>><490 nm.
>>
>>Before sacrificing any animals, I strongly recommend testing your
>>vector,
>>and practice the DNA counterstain, on tissue culture cells. If you
>>have an
>>inverted scope, grow the cells in the mattek dishes, infect, then
>>look at
>>the cells live (optionally add DAPI or Hoechst, best to use phenol
>>red free,
>>low serum or no serum media during the imaging).
>>
>>
>>Best wishes,
>>
>>
>>George
>>
>> -----Original Message-----
>> From: Caroline Bass [mailto:[log in to unmask]]
>> Sent: Saturday, November 19, 2005 1:16 PM
>> To: [log in to unmask]
>> Subject: counterstain for soluble EGFP
>>
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> Hello,
>>
>> I am attempting to characterize a viral vector. Right now I
>>am
>> injecting a virus that makes soluble EGFP and collecting the
>>liver,
>> sectioning and taking photos of the native fluorescence. I
>>have a
>> great signal but I am having a difficult time determining
>>which cells
>> are positive. I would like to characterize which cells are
>>
>> transduced and get a general idea of the percentage
>>transduced. The
>> easiest way to do this is probably DAPI staining I think,
>>just to get
>> an idea of the number of cells and possibly determine the
>>percentage
>> transduced. Could someone recommend anything that will help
>>me
>> determine which cells are staining? I am relatively new to
>>
>> microscopy especially fluorescence. I would love something
>>that I
>> can visualize easily on a standard fluorescent microscope as
>>well.
>> Could I do an H&E staining on the sections and still see the
>>
>> fluorescent signal? If not is there an alternative? I
>>would love to
>> see a typical stain that would highlight the various cell
>>types and
>> then overlay my fluorescent signal in the same field.
>>
>> Any and all advice is appreciated.
>>
>> Sincerely,
>>
>> Caroline Bass
>
>
>
>George McNamara, Ph.D.
>Division of Cancer Immunotherapeutics and Tumor Immunology
>City of Hope National Medical Center
>1500 E. Duarte Rd
>Duarte, CA 91010
>626-359-8111 ext 60035
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