Re: RE: GFP visualization LONG reply

I'm beginning to work with EGFP mice and I've been reading 
contradictory stuff regarding the fixation of frozen tissue. Any help? 
Thanks. Ouahiba

----- Original Message -----
From: "Montague, Donna C" 
Date: Monday, February 24, 2003 10:20 am
Subject: RE: GFP visualization LONG reply

> Ben and interested others:
> Ben asked:
> --"Due to potentially low signal, we believe a biotin-avidin-HRP 
> system is necessary for our application, as direct observation 
> doesn't seem feasible.We are attempting to analyze rat stromal 
> cells expressing GFP, so I wanted to follow-up on any suggestions 
> you might have to avoid non-specific binding since the antibody is 
> mouse anti-GFP. 
> 1.)     I also read where you use hydrocarbon solvents.  We use 
> Xylene here, so I was wondering if you believe this one difference 
> could affect our results?
> 2.)     Do you perform any antigen retrieval for the Zymed enzyme?
> 3.)     Have you observed low background in GFP negative controls?
> 4.)     Is this Zymed MAB the best to use, in your opinion?  Other 
> options?5.)     How long do you incubate in primary and at what 
> temperature?6.)     In what diluent?
> 7.)     What blocking buffer might you recommend?"
> GFP visualization in paraffin processed tissue can be tricky. The 
> following "pearls" are taken from my own experience and reflect 
> what we do in our lab. Neither the University of Arkansas for 
> Medical Sciences nor I have financial interest in the companies 
> whose products are mentioned in this email. That said, this is 
> what we do.
> Given that we are a full-service research support laboratory and 
> receive specimens from different sources over which we have little 
> control, we try to ascertain which GFP product (wild-type (wt) or 
> variant (e.g. EGFP)) was used in the specimen. If wtGFP was used, 
> we process the specimen for paraffin as usual and then attempt to 
> visualize the GFP using an antibody. We have used both Zymed's 
> antibody and Clontech's. Even though they are recommended for 
> western blot, they work well in paraffin. We start with a 
> concentration 10 fold less than that recommended for western blot 
> (e.g. if the WB recommended concentration is 1:1000, we would 
> start with 1:100 and probably bracket with 1:50 and 1:200 for the 
> primary). 
> GFP tags may be found anywhere within the cell, attached to the 
> inside or outside of the cell membrane or extracellularly 
> depending on the product to which they are attached. Therefore, 
> some permeabilization of the cell membrane may be required. Our 
> routine procedure for new antibodies is to:
>    1) Fix well in 10 % neutral buffered formalin
>    2) Decalcify with Formic acid or EDTA as appropriate
>    3) Dehydrate in serial alcohols starting with 70% through 100 
> % until all fat and water have been removed
>    4) We clear with methyl salicylate for bone and xylene for 
> soft tissues.
>    5) We infiltrate with a 50:50 mixture of EM400 (Surgipath) and 
> Tissue Prep 2 (Fisher) using three changes
>    6) We embed in 100 % EM400
>    7) Cut at 5 um
>    8) Float onto SuperFrost Plus (for immuno) or UltraStick (for 
> IS-PCR).
>    9) Anneal with heat and allow to cool flat.
>    10) Deparaffinize to dH20.
>    11) Immerse in PBS (pH 7.4) containing 0.02 % Triton X-100
>    12) Antigen retrieval (Biogenex Antigen Retrieval Citra 10X 
> concentrate, cat # HK086-5K) is accomplished by immersing the 
> slides in 80 - 90 oC citrate buffer (pH 6.0) for 15 minutes. We 
> never microwave the slides in the buffer but rather heat the 
> buffer then place the slides in the hot solution. Recall that 
> tertiary and quaternary proteins unfold at temps less than 95oC. 
> Steaming or boiling the slides is merely asking for the tissue to 
> fall off the slide regardless of the adhesion additive used.
>    13) Slides are rinsed in PBS-Triton
>    14) Block for endogenous peroxidase activity using 3 % H2O2 in 
> PBS-Triton or Methanol as you wish. Rinse in PBS-Triton.
>    15) Apply 1 - 5 % host (e.g. if the antibody was made in 
> mouse, use mouse serum) serum in PBS-Triton to block non-specific 
> binding. (If non-specific staining is still a problem use a 
> completely different species, e.g. horse, donkey or goat).
>    16) Apply the primary antibody and incubate at 4 oC overnight 
> or at 25 oC for 1 - 3 hours.
>    17) Rinse with PBS-Triton times 2 for 5 minutes each.
>    18) Apply secondary antibody, incubate according to package 
> insert usually 20 minutes at room temp is sufficient
>    19) Select and develop chromophore as desired.
> For additional info on GFP see the FAQ section of the Histotech 
> Home Page at  <" target="l">> 
> Hope this helps.
> Donna Montague
> University of Arkansas for Medical Sciences
> Department of Physiology & Biophysics and
> Center for Orthopaedic Research
> 4301 W. Markham St. # 505
> Little Rock, AR 72205

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