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Author Topic: Warp red chromogen staining the slide itself.  (Read 2532 times)

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Offline BVirginia

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Warp red chromogen staining the slide itself.
« on: May 04, 2015, 10:30:10 AM »
Hi! Im an employee of the Molecular Pathology core lab here at Yerkes National Primate Research Center in Atlanta GA. We do primarily IHCs, IFs, ISH/FISH, etc. My line of questioning centers around the Biocare Medical Warp Red chromogen.
We have been seeing this pinkish hue on our slides. Not every antibody causes this pink hue we mostly have seen it with lysozyme antibodies (for which we use Diva Decloaker antigen retrieval solution) and with an influenza A antibody that requires antigen fixation for 2 hours in acetone instead of the usual HIER.
I have attached some images of these affected slides so you can see what it looks like.
I have also talked to the Biocare Medical technical support a couple of times about this already, and the consensus was that our issue was caused by non-sterile water or insufficient rinses.

Some of the steps weve already tried to get rid of chromogen to slide adhesion.
1)   Baking for 2 hrs at 60C and then immediately beginning deparaffinization to improve paraffin removal.
2)   A variety of bloxall and protein block combinations.
3)   Cleaning the water bath with bleach once a week and with 70% etOH after each use.
4)   Immersing slides in TBS-T between antibody incubations as an extra rinsing step, i.e. rinse off the reagent, then immerse slide in TBS-T for about 2 min.
5)   Using sterile, purchased water to prepare reagents and for rinsing slides, rather than water from a deionization/sterilization unit.
6)   Trying out different brands of slides to see if its the Fisher Superfrost plus charged and coated slides that we are using.

Thank you for your help!

Warp red chromogen staining the slide itself.
« on: May 04, 2015, 10:30:10 AM »

Offline gula

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Re: Warp red chromogen staining the slide itself.
« Reply #1 on: May 04, 2015, 01:20:05 PM »
It may be, that you activate binding sites on the charched slides by the special pretreatments. Have you tried to stain a blank slide after such treatment just in chromogen?
The paraffin can still be seen on some slides. Are these "ghosts" gone after better deparaffination?
gula

Re: Warp red chromogen staining the slide itself.
« Reply #1 on: May 04, 2015, 01:20:05 PM »