Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
ALong with what George said, the Hg lamp used for widefield fluorescence
has a very strong peak ~546nm, and no corresponding peak for the
excitation range used for GFP.
Cheers,
Jeff
> -----Original Message-----
> From: George McNamara [mailto:[log in to unmask]]
> Sent: Sunday, November 20, 2005 7:42 PM
> To: [log in to unmask]
> Subject: Re: counterstain for soluble EGFP
>
> ---------------------- Information from the mail header
> -----------------------
> Sender: Confocal Microscopy List <[log in to unmask]>
> Poster: George McNamara <[log in to unmask]>
> Subject: Re: counterstain for soluble EGFP
> --------------------------------------------------------------
> -----------------
>
> Search the CONFOCAL archive at
> 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
> >[log in to unmask]
> >[log in to unmask]
> >
>
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