--"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
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
5.)How long do you incubate in primary
and at what temperature?
7.)What blocking buffer might you
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
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).
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
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
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
6) We embed in 100 %
7) Cut at 5 um
8) Float onto SuperFrost Plus (for
immuno) or UltraStick (for IS-PCR).
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
13) Slides are rinsed in
14) Block for endogenous peroxidase
activity using 3 % H2O2 in PBS-Triton or Methanol as you wish. Rinse in
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