[Histonet] troubleshoot of positive controls for PKCalpha and eNOS antibody

[shazana hilda]

Dear Histonetters,
 
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 <@t> 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 few journals for positive controls of both antibody. however my positive controls 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 <@t> yahoo.com