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

From:"Patsy Ruegg"

In my experience drying even after DAB is not good at least in some cases
the tissue does not far well and the morphology can be adversely affected.

-----Original Message-----
From: ihcrg-bounces@neo.agsci.colostate.edu
[mailto:ihcrg-bounces@neo.agsci.colostate.edu] On Behalf Of Ni, Ruoyu
Sent: Thursday, February 24, 2005 7:26 AM
To: 'Patsy Ruegg'; leharkins@yahoo.com; TJJ@stowers-institute.org;
Cc: histonet@lists.utsouthwestern.edu
Subject: RE: [IHCRG] Automated immunostainer for research applications

I think, at this point(after DAB and washing), it does not matter if the
slides are dry or not.
Ruoyu Ni, MLT
Molecular Histology
Mount Sinai Hospital
Toronto, M5G1N6

-----Original Message-----
From: Patsy Ruegg [mailto:pruegg@msn.com] 
Sent: February 24, 2005 9:12 AM
To: leharkins@yahoo.com; TJJ@stowers-institute.org;
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [IHCRG] Automated immunostainer for research applications

I use the DAKO autostainer overnight somewhat different from Loui.  I set 
the run to go with usually a 1-2 hr. incubation of primary and complete run,

my last wash is dih20 so after the run finishes the autostainer will apply 
dih20 once every hour until I end the program the next day, this way I do 
not waste buffer as after the run finishes dih20 can be applied to keep the 
sections from drying out.

Patsy Ruegg, HT(ASCP)QIHC
IHCtech, LLC
Fitzsimmons BioScience Park
12635 Montview Blvd. Suite 216
Aurora, CO 80010
cell 720-281-5406
wk email pruegg@ihctech.net
hm email pruegg@msn.com
web site www.ihctech.net

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----Original Message Follows----
From: lualhati harkins 
To: "Johnson, Teri" , 
CC: Histonet 
Subject: Re: [IHCRG] Automated immunostainer for research applications
Date: Wed, 23 Feb 2005 18:12:23 -0800 (PST)

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 microscope?

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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