I get very high staining with hematoxylin. I want to reduce it. How can I possibly reduce it? I use Ammonium hydroxide and water washing steps afterwards.
You can either dilute it as suggested or dip/apply to your slides for a shorter period of time. Also, you can use less intense formulations: Mayer's vs. Harris' haematoxylin. I recommend practicing different conditions on some inessential slides; you'll figure out what works via trial and error.
I would suggest using Harris' hematoxylin and immerse the slides for 2 minutes. Blue in tap water or tap water subsitute like Scotts and then differentiate in acid alcohol for a few seconds. this is made from 70% alcohol and 1% HCL. Then re blue in tap water. you can have a look down the micrscope before you dehydrate and mount to ensure you have the right level of staining; too much, pop them back the the acid alcohol, to little, restain in the hematoxylin but don't differentiate as much.
For immuno on paraformaldehyde fixed specimens we use standard Harris's for around 6 seconds to achieve light nuclear staining that is a nice contrast to either DAB or AEC chromagen
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