[Histonet] RE: [IHCRG] Automated immunostainer for research applications

From:"Patsy Ruegg"

As for antibody amount.  On the DAKO depending on the size of the tissue I
use 100 microliters to 200 microliters, I always use a pap pen to isolate
the reagent over the section, I put all my sections at the bottom of the
slide as the reagent tends to draw up towards the label, I just draw one
line straight across the slide just above the section at the bottom to keep
the reagent down at the bottom over the section, I deparaffinize, rehydrate
to 95% alcohol and then let the sections dry, I then draw the pap pen line
while the section is dry before rinsing with dih20 and then buffer, some pap
pens these days even survive HIER (BioCare's does), I have even noticed that
this one is still there after passing thru alcohols and xylene to coverslip,
others I have used will not survive the solvents.
As Loui, if I am developing a protocol, I check the DAB development
microscopically until I get an optimized time, I then program the machine
for that time for that protocol.
I am an advocate for longer incubation times with more dilute antibodies, I
feel you get cleaner staining and more efficient use of the antibodies.  I
also dilute detections and use them for longer periods in this same way.  I
know in clinical practice everybody is in a hurry but I think you pay in
cost of reagents and signal/noise by using antibodies too concentrated for
short times.

-----Original Message-----
From: ihcrg-bounces@neo.agsci.colostate.edu
[mailto:ihcrg-bounces@neo.agsci.colostate.edu] On Behalf Of lualhati harkins
Sent: Wednesday, February 23, 2005 7:12 PM
To: Johnson, Teri; ihcrg@neo.agsci.colostate.edu
Cc: Histonet
Subject: Re: [IHCRG] Automated immunostainer for research applications

Hi  Teri,
In response to your first question on overnight incubation( automated
1) Overnight incubation is achieved on  Biogenex I6000 , with Optimax and
also with DAKO Autostainer through setting up the program on delayed run.
The deparaffinized slides are loaded into the machine and set on continious
buffer run to prevent slides from drying up.
Antibody incubation time- i optimize my antibody dilution to allow 1-2 hours
of incubation at room temperature.This allow me to use very low dilution of
primary antibody which answer your other question:
2)" what if you have only 500 microliter of antibody available to use"
I was able to use 200microliter ( Biogenex i 6000or Optimax) or less than
200 in some instances with Dako Autostainer.
If you do not have any time as well as enough antibody for optimization:
 a ) find supporting data such as : dilution used for Immunofluorescence- (
frozen)- By previous experience i found out that if immunofluorescence
working concentration (frozen) is 1:500
then i will use 1:50 as dilution for paraffin sections
b) if i still do not have enough antibody to come up with this dilution,
then i will use 1:100 and run an overnight incubation in the refrigerator.
c) western blot data is useful also: if westernblot working concentration is
1:500 , then start a working dilution at 1:50 for paraffin sections, or if
using frozen then use 1:250 ( this will really need some optimization
For overnight incubation, start with 1:100 for paraffin sections, frozen
sections could possibly work at 1:500 overnight, but not a sure thing; I
have better luck for sure signal to stay around 1:300.( these are just
This dilution gets complicated depending on how much protein concentration ,
and the type of protein and also where the localizationis expected :
nuclear, cytoplasmic/membrane. 
Please contact me directly for detailed and in depth discussion of other
data determining dilution.
To prevent  using too much buffer, i also adjusted my secondary antibody
and HRP label
incubation time for 45 minutes each.
With respect to chromogen use, for research purposes, i prefer to develop
chromogen and observe signals through te microscope. For diagnostic runs, i
optimize the chromogen development to run in the machine for puposes of
other personnell  using the machine when i am on vacation
Finally to give an example of overnight/delayed run in the machine:
Start machine at 6Pm
Delay-(4hrs)  -start at 10PM
Antibody Incubation- done at 12AM
Secondary/label done at 1:30 AM
Buffer rinse until morning- 
You can adjust the delay for however long you want-
I hope the information will be useful.
I hope i was able to help you

"Johnson, Teri"  wrote:
Hi all (and apologies to those on both lists),

For those of you doing research histology and immunohistochemistry,
which automated immunostainer do you use? Do you do any overnight
incubations with it? For those of you who have automated stainers and
formerly did overnight (4C) incubations, what modifications did you make
to your staining protocol to achieve the same result? Also, do you
always use the substrate on instrument, or do you do that via

Some immunostainers require minimum amounts of primary antibody (~500
microliters), even if staining only one slide. Some times we don't have
that much antibody to use. Are there any tips to get around this with
the exception of manually pipetting as if doing a titration run?

Thanks for your input!

Teri Johnson
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, Missouri 64110

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Please note: Protocol and technical information provided by the undersigned
are developed on site with conditions limited to fixation of
tissues,reagents,control tissues and execution of protocol which is
characteristic only of our laboratory and therefore we do not bear neither
responsibility nor liability for results outside of our laboratory. It is
our courteous gesture to share
information that we deemed might be useful to laboratorians and
Loui Harkins , M.S. QIHC
Biologist, Immunohistochemistry/Path.& lab. Medicine
DEpt. Of Veterans Affairs,700 South, 19th Street
Birmingham, Alabama 35233
(Research Consultant) Dept .of Surgery, Neurosurgery Division
University of Alabama in Birmingham
205-933-8101 ext.6719; fax 205-558-4817 or 558-7034
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