troubleshoot for positive control of PKCalpha and eNOS antibody by IHC method

[hilda]

Dear IHC members,

I'm having difficulties to obtain positive staining of positive control for PKCalpha (H-7:sc-8393) and NOS3(c-20:sc-654) antibody. Both antibody were purchased from SantaCruz Biotechnology Inc.and used for IHC.Below are the methods that i used:

1)PKCalpha

    -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

2) 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 both 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]

The suggested dilution for eNOS is 1:500 (NOT 1:50). In some occasions, higher dilution gives better staining than lower dilution, especially for certain rabbit antibodies. So try 1:500 dilution and let us know the result.
 
Also try to stain eNOS without antigen retrieval as some of eNOS antibodies I worked with showed good staining with NO antigen retrieval condition.