RE: Insulin-like growth factor I antibodies on mouse tissue

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From:Patsy.Ruegg@UCHSC.edu
To:JErickson@genetics.com
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I have used anti-human IGF-I and IGF-II successfully on rat tissue embedded
in paraffin, from Intergen, with a brief (4 min.) pretreatment with
proteinase k, not hier.  I fix with zinc formalin.
Patsy Ruegg

-----Original Message-----
From: Jamie Erickson [mailto:JErickson@genetics.com]
Sent: Thursday, February 17, 2000 8:51 AM
To: HistoNet@pathology.swmed.edu
Subject: Insulin-like growth factor I antibodies on mouse tissue


Hi All,
	           I was wondering if anyone has experience with antibodies
to insulin-like growth factor I (IGF-I) for use on mouse tissues, I have two
antibodies.  One is from Santa Cruz a goat polyclonal the other from
Intergen is  a rabbit polyclonal. I have tried these on mouse tissues Frozen
(various [conc]) and paraffin with little luck. My results were that the
antibody would stain mouse uterine tissue a  possible  positive control but
would not dilute out and the rabbit IgG Polyclonal antibody I used as a
isotype control was also staining the same way and not diluting out. I
should also state that I used both  C57Bl/6 and C3H/Hej mouse uterus as
positive and negative controls, respectively ( data from investigator ). The
paraffin samples showed no staining so at this time I would just like to get
the IGF to work with no background in the frozen samples, the spec sheets
says  it works in ELISA and Westerns but as usual no data on IHC. I'm at my
wits end with this stupid antibody it should have worked on my routine
screening, so if you have other antibodies  that work or have any experience
with this or tricks like blocking etc..please contact me by e-mail or phone.
Below is my standard protocol,  I have to get this working to finish this
project and  get my supervisor off my back please help. Thanks for any
help...

Test antibodies were diluted to final concentrations ranging from 1mg/mL to
20 mg/mL.  All test samples  were selected for IHC evaluation for the
presence and localization of IGF-1. Prior to staining, the paraffin-embedded
slides for IGF-1 staining were enzyme digested with proteinase K (25 mg/mL)
or Trypsin (Pre-diluted). Cryopreserved samples were sectioned at 5 mm onto
glass slides, fixed in cold acetone at -20C for 1 minute, and then stored
at  -20C until time of staining. Immediately prior to staining, stored
cryosections were equilibrated to room temperature, fixed in cold acetone
for 10 minutes, air-dried, blocked with avidin/biotin block (Zymed
Laboratories, Inc., South San Francisco, CA) for 10 minutes each, as stated
in manufacturer instructions, and washed in PBS. 

Next the slides were assembled on the Ventana TechMate 500 automated
immunostainer and stained using a program for cryopreserved or fixed tissues
(Notebook # 6092, pages 3, 4 and 151). Using this system, sections were
exposed for 1 hour to either primary antibody or an appropriate isotype
control (Rabbit IgG), reacted with a biotinylated secondary antibody
directed against the test antibody host immunoglobulin for 30 minutes,
followed by an streptavidin peroxidase linker (Signet Pathology Systems,
Inc.). Sections were then incubated with the chromogen 3, 3'
diaminobenzidine tetrahydrochloride (DAB) counterstained with hematoxylin,
dehydrated to xylene, and coverslipped with synthetic mounting media. 

The protocol for cryopreserved tissues was used (see notebook # 6092, pages
3-4) with some modifications, which included the following: 1.) the addition
of  5% horse serum to the blocking buffer, as well as to the rabbit and goat
primary antibodies respectively  2.) 12.5% hematoxylin was used to
counterstained the slides, 3.) the incubation in primary antibody was
changed from 30 minutes to 1 hour, and 4.) the blocking buffer time was
changed to 15 minutes. 



Jamie Erickson
Associate Scientist
Genetics Institute
1 Burtt Rd.
Andover, MA   01810
work : (978) 247-1348
FAX  : (978) 247-1333




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