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Author Topic: ***Osteocalcin - Problem with DAB and Hemtaoxylin***  (Read 2869 times)

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Offline supcali

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***Osteocalcin - Problem with DAB and Hemtaoxylin***
« on: July 17, 2009, 12:22:14 AM »
Hi,

I am doing an Osteocalcin staining on Paraffin-embedded sections with:

- 30 min Antigen Retrieval @ 37 deg
- 45 min peroxide @ Room Temp
- 20 min blocking @ Room Temp
- Overnight primary Ab @ 4 deg,
- 45 min Secondary Ab @ Room Temp
- 8 min DAB followed by one 5 min wash in DI water (I don't change the solution of water here)
- 30 sec Hemtaoxylin followed by quick dip in DI water and then 3 rinses with PBS (first two rinses are 10 dips and last rinse is for 5 min)

- I then dehydrate slides through increasing concentrations of EtOH (25-50-75-100) then two washes of Histoclear (5 min each)
- Mount with Cytoseal

My staining looks horrible!

- There is a lot of non-specific DAB staining
- Whichever tissue had DAB staining (+primary Ab) there is NO hematoxylin. Tissue without DAB (- primary Ab) has faint hematoxylin

I am using Sigma Mayer's Hematoxylin and DAB from Vector.

PLEASE HELP!!!!
« Last Edit: July 17, 2009, 12:34:58 AM by supcali »

***Osteocalcin - Problem with DAB and Hemtaoxylin***
« on: July 17, 2009, 12:22:14 AM »

Offline excalibur

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Re: ***Osteocalcin - Problem with DAB and Hemtaoxylin***
« Reply #1 on: July 17, 2009, 10:42:10 AM »
Increase the blocking time.

I never block with 3% H2O2 for more than 10 min.

Are you rinsing after the primary and secondary? (Assume your secondary contains both link and label)

I rinse with tap water after hemat to blue it. Don't waste your PBS.
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
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Offline supcali

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Re: ***Osteocalcin - Problem with DAB and Hemtaoxylin***
« Reply #2 on: July 17, 2009, 12:51:38 PM »
Yes, I am washing after primary and secondary.
After primary, I wash 2x 2min with PBS. After secondary, I was 3x 5min with PBS.

Any other suggestions?

Thanks

Re: ***Osteocalcin - Problem with DAB and Hemtaoxylin***
« Reply #2 on: July 17, 2009, 12:51:38 PM »