Problems with FluoroJade staining

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Problems with FluoroJade staining

Post by fabulousparrot » Mon May 20, 2013 1:47 pm

Hello everyone,

I am trying to stain degenerating neurones with FluoroJade B. I have no training in histology, so excuse if something is out of place.

The sections swallow and wrinkle and some of them even get out of the slide. It's typically perfused brain slices, preserved in antifreeze at -20C as a free floating section, washed in PBS, mounted in slide (super frost, positively charged) and dry overnight.
This happens at step 3 of the protocol, I will slash all the variations I've found on the internet and tried with no success:

1) 5 min 80% ethanol, 20% NaOH/ 5 min 100% ethanol/ 3 min 80% ethanol
2) 2 minutes 70% ethanol/ 1 min 70% Ethanol
3) 1 minute dH2O (here is where the wrinkles start to show up)

the rest of the protocol is as it follows:

4) 15 minutes in 0.06 % potassium permanganate (by then tissue might be already floating or only half attached to the slides)
5) 2 min wash in dH20
6) (From here everything away from light as possible) 30 minutes in FluoroJade solution 0.0004%
7) rinse in 3x1minute of dH20
8) dry overnight
9) one minute in xylene, DPX and dry

My questions are:

A) Should I rehydrate the slides slower than in the protocol (but why would anyone succeed then?) doing ethanol progressions? I wonder why would you dehydrate the samples to put them into a minute of dH20 water in step three… Can I skip step 1 and 2?
B) Can I use clearing agent before drying? Or can I substitute drying by dehydrating by ethanol baths? I also wonder if I can substitute xylene by histoclear?
C) Why are my sections getting bubbles and floating?

Thanks a lot, folks!

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