Re: [Histonet] IHC for PECAM-1

From:Angela McNabola

Hi Francesca,

We have used this antibody on several xenographs (including PC3) with nice
results.  Incidentally, this is the only PECAM-1 (M20) from SC that we have
gotten to work in FFPE tissues.

We do it on an automated stainer, but it can be done offline as well.

Here is our protocol:

1.    5um paraffin sections were deparaffinized in xylene and brought to water
through a graded series of alcohols.
2.    Antigen retrieval was performed using Dako Target Retrieval Solution
(DakoCytomation, S1699, Carpinteria,CA).  Buffer was prepared according to
package insert.  Slides were placed in heated buffer and steamed (in a
pre-heated Black and Decker steamer) for 30 minutes, then cooled for 20 minutes
at room temperature.
3.    Slides were rinsed in Dako wash buffer (S3006, DakoCytomation,
Carpinteria, CA) and then placed in 3% hydrogen peroxide (in water) for 5
minutes to block endogenous peroxides.
4.    Slides were rinsed with Dako wash buffer and an avidin /biotin block was
performed using an Avidin/Biotin Blocking kit (Vector Laboratories, Inc.,
Burlingame CA, cat# SP-2001).  Rabbit serum was used as the protein block (30
minute incubation), and the Avidin and Biotin were incubated for 10 minutes
5.    Immunohistochemistry was performed using a goat Vectastain ABC elite kit
(Vector Laboratories, cat # PK-6105) following package insert beginning at the
primary antibody step with the following modifications:  two thirty minute
incubations were performed with primary antibody and following the incubation of
the secondary antibody slides were rinsed in buffer, distilled water, and buffer
again.    Primary antibody used with the kit was CD31 antibody (PECAM-1 (M-20)
SC-1506 goat polyclonal, 200 ug/ml, Santa Cruz Biotechnology) at 1:750 (using
Dako Antibody Diluent – cat# S0809, DakoCytomation, Carpinteria, CA). Goat IgG
(1:750) ( code# 005-000-003, Jackson Immuno Research Laboratories, INC., West
Grove, PA) and rabbit serum (protein block) were used as negative controls.
DAB was the chromagen used (DakoCytomation., Carpinteria Ca.).
6.    Slides were counterstained with Hematoxylin (Automation Hematoxylin,
S3301, DakoCytomation, Carpinteria,CA).

                      "Francesca Wannenes"                                                                                                    
                      t>                                    cc:                                                                               
                      Sent by:                              Subject:  [Histonet] IHC for PECAM-1                                              
                      04/21/2004 06:54 AM                                                                                                     


I want to test the PECAM-1 expression in PC3 xenograft tumors.

I have the primary antibody PECAM-1 (M20) and the ABC staining system of Santa
Cruz Biotechnology.

My samples are paraffin-formalin embedded.

Have you a method to use these antibodies for the immunohistochemistry? If not,
have you similar methods for immunohistochemistry for to test CD31 (PECAM-1)

Thank you to everybody

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