Hi, there
We are having some trouble trying to combine immuno-based colorimetric assay and methylene green staining, since we perform the inmuno as usual and then when we try to get methylene green staining, it washes off inmediately at either water or 70% ethanol. We've tried leaving it in methylene green for as long as 12min but it still goes away and we've tried to skip water and go straight to 70% ethanol and it didn't work either.
We don't understand what could be the problem here since normal methylene green staining (meaning, with no inmuno beforehand) works just fine in the same kind of paraffin sections and same kind of slides. Paraffin sections are of 7 uM thick.
If anyone has a suggestion or knows what could be the issue here we woul'd be very thankful
Thank you very much in advance
Hallo Beatriz, Methylene green is not a real suitable counterstain. The reason for the lost staining ability after immun could be the pH, or defferences in ultrastructure of the tissue when you have used detergents like TritonX-100 or Tween or when you have done any kind of heat induced antigene retrievel.
If you used DAB (brown) as a chromogen to visualize your antigen-antibody reaction Hematoxylin (blue) is the most recommanded one. If you use Nickel enhancement (black blue colour) then Nuclear Fast Red (red) is recommanded.