RE: GFP long reply if not interested delete now

<< Previous Message | Next Message >>
Content-Type:text/plain; charset="iso-8859-1"

Pravin and interested others:
You stated: "I have frozen sections of bone marrow and I am trying
to detect GFP fluorescence.  I dapi fluorescent mount
them and everything glows in there.. Any suggestions
on how to get rid of the autofluorescence and let the
GFP shine through.."
1. Which GFP clone are you using as your reporter? If you are using EGFP,
you can fix the frozens using acetone, if you are using other (wild type GFP
reporters) you need to use formalin to fix the slide.
2. Do you really need dapi to see the nuclei? Probably not, there is
significant bleed over between dapi and GFP emission lines. I assume you're
using a standard fluorescent microscope. Do you have access to a Confocal
microscope? The use of laser light by confocal microscopes significantly
decreases emission line bleed over. If not, omit the dapi and view the
section. GFP should provide you with enough light to discern the cell.Also
use a good quality mounting medium such as Vectashield Vector Labs #HT-1000
3. Autofluoescence of bone marrow sections can be decreased by a couple of
tricks: (a)thinner sections lead to lessened "out-of-focus" contributions of
the autofluorescent porphyrins (hemaglobin and the like). (b) treat the
section with 0.1 % acetic acid in PBS + 0.02% Triton X-100. About 3-5
minutes followed with a PBS-Triton wash.

Your next query: "I have been trying to work out a IHC protocol
using the mouse monoclonal from Clontech and have a
good deal of problems with background binding.. Do you
know of any published info, or do you have any advice
on some good techniques.."

I have a comparative paper on GFP techniques just about ready for
submission. I'll be presenting the summary and techniques in my workshop at
NSH this fall (workshop # 95). But for IHC, I've had better luck with the
Zymed antibody to GFP (Zymed # 33-2600)at a dilution of 1:1000 in
formalin-fixed paraffin embeded (FFPE) formic acid decalcified bone
sections. It is also a mouse monoclonal, so if your specimen is mouse or rat
you'll need to modify your immuno protocol to decrease the non-specific
binding and enhance the specific staining.
 A suggestion: Prepare your sections to distilled water. If formalin fixed,
perform a HIER method such as immersion of the slide into 75-80 oC 0.1 M
citrate buffer (pH 6.0). Warning: do not put bone sections into a room temp
solution and heat them up with the buffer to near boiling. The sections will
fall off the slide regardless of the slide adhesive (nearly, I'll comment on
that later). Rinse the slides in PBS-triton. Prepare your primary antibody
as follows: in a 1.5 mL eppendorf tube, place 984 uL of PBS-triton, add an
aliquot of the primary antibody to yield the appropriate concentration e.g.
1 ul mouse monoclonal anti-GFP (Zymed # 33-2600). Add 5 ul of biotinylated
anti-mouse IgG and let set at room temperatrue 15 minutes. Add 10 ul of
normal mouse serum let set 5 minutes then apply to your sections.Detect as
desired using a strepavidin conjugated chromagen/fluorochrome.

Hope this helps. Let me know how it turns out. Any other suggestions out

Donna Montague, Orthopaedic Research, University of Arkansas for Medical
Sciences, Little Rock, AR, USA

<< Previous Message | Next Message >>