troubleshoot for positive control of eNOS antibody by IHC method

[hilda]

Dear IHC members,

I'm having difficulties to obtain positive staining of positive control for endothelial NOS3(c-20:sc-654) antibody. antibody was purchased from SantaCruz Biotechnology Inc.and used for IHC.Below are the method that i used:

1) NOS3@eNOS

    -incubation time: 1hour,room temperature (24-25 celcius)

    -Antibody dilution: 1:50

    - Target Antigen Retrieval Buffer: Citrate Buffer, pH6

    -Device: microwave (3x at 600W for 5min)

    -Detection method: polymeric method by LabVision

I've used normal rat kidney as suggested by IHC WORLD protocol for positive controls of antibody. Yet, my positive controls still produced negative staining.

Your suggestion and quick response are highly appreciated
 

Shazana Hilda Shamsuddin
MSc.of Molecular Pathology,
Dept. of Pathology,
School of Medical Sciences,
Universiti Sains Malaysia,
16150 Kubang Kerian,
Kelantan, Malaysia.
Email: hilda_1075@yahoo.com
 

[richard03]

Try to stain eNOS without antigen retrieval step as some of eNOS antibodies I worked with showed good staining with NO antigen retrieval condition.