Jacqui,

Methyl green does not like water, especially after the slides have been treated by HIER and immunostained. Side-by-side slides with & without pretreatment show good methyl green counterstaining without, and poor with.

You can try using acetone to rinse and dehydrate your slides after staining. We find that works well, but it's very messy.

Alternately, you can try using a 10% solution of hematoxylin for 30 seconds to counterstain, and it should keep the nuclei from getting too dark. If it's still too dark, adjust your time accordingly. If you're using AEC, be sure you don't use an alcoholic hematoxylin, or you run the risk of losing your positive signal.

Best wishes,

Teri Johnson
Stowers Institute for Medical Research
Kansas City, MO 64110

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