Insulin-like growth factor I antibodies on mouse tissue

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From:Jamie Erickson <JErickson@genetics.com>
To:HistoNet@pathology.swmed.edu
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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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