[Histonet] DAB & tissue adherence issues on rabbit bone tissue

[Wilson, Lindsay A]

Good morning,

I am currently experiencing an issue with DAB concentrating on certain bone areas of my slide. I am working with decalcified—EDTA , FFPE- rabbit mandible (some tissue attached). It is a new DAB kit from BioCare. It’s thoroughly mixed (and new) before application in a humidity chamber with the slides lying flat.

I change out the deparaffinization soluitons frequently, my reagents are in date, I have increased the peroxidase time to 10 minutes, I use a blocking reagent from Millipore kit  for 5 minutes (manuf. Recomm.), 2.5% goat serum for 20%, my wash buffer is 1x TBST with 0.05% Tween 20.

The FFPE slides are aired dried before use. I have seen posts about baking them in the oven afterward – that doesn’t affect the slides at all?

I also have the issue of the sample falling off and/or moving on my slide. The slides are positively charged, I am currently performing AR with an AR ph6 buffer (for VEGF antibody) in 95C microwave for 15 minutes. I have previously tried a 60C waterbath for 12 hours with the same issue.

I am thinking that the issues are connected? If the tissue isn’t moving around or falling off, perhaps the excessive DAB wouldn’t be an issue? I have seen posts about the Biogenex DeCal solution – is that an effective method?

I can attach images if needed.

Lindsay Wilson, LVT, RLATG
Young Laboratory

UTHealth | The University of Texas Health Science Center at Houston | School of Dentistry
Department of Oral & Maxillofacial Surgery

7500 Cambridge St. | Suite 6510 | Houston, TX 77054
713 486 4360 tel | 713 486 4333 fax