RE: GFP visualization LONG reply

From:"Montague, Donna C"

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
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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