I inherited a staining protocol from another lab. It specifically says not to swirl the ABC mixture, but instead to let it sit for 30 minutes before adding the tissue. It does not specify if the tissue should be agitated while incubating in ABC. I usually always agitate because my sections are large and therefore fold on themselves somewhat and I want to ensure adequate circulation of the reagents around the tissue. I'm wondering if anyone knows whether ABC should never be mixed/swirled/agitated or if that only applies to the initial addition of reagents to the buffer.
From a purely biochemical standpoint, I would assume you would want the A&B components in constant motion as there is no directed binding; it's purely stochastic. The kit says to mix well initially and let stand, but there's functionally no difference between rocking and standing if the two components are well mixed.
For my own protocols, I have added the A&B components, inverted a couple times, agitated on a seesaw rocker for an hour before adding to my tissue and it works fine. I have used the ABC elite kit on floating sections and slides with no issue. The solution is saturated by design and as long as your sections are covered, you should see signal. I definitely advocate a gentle motion for free floating sections due to the potential folding of the tissue.
I have always used a belly dancer when using the kit with CNS tissues and never had any problems!
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