In our lab we have procured fluro jade-B from Millipore. We have tried many times using the protocol given with the product. Still we didn't get any expression. If anyone has done the same please give me some suggestions.
Hi Kaliappan, time window is critical for Fluoro Jade-B staining because Fluoro Jade is used to label dying or dead neurons in brain tissues. If you collect and fix tissues too early or too late, you have fewer chances to get positive staining. This is not a complex technique. Make sure you have dead/dying neurons in your tissue samples using other staining techniques such as Cresyl Violet. What neurodegenerative model are you working with?
BTW, what do you mean by “did not get any expression”?
You can follow the following step for the tissue section attached in the slide glasses
1. warm the tissue for about 30 min at 55 degree slide warmer
2. wash with water for few seconds
3. wast with 100 % EtOH for 3 min followed by 70% EtOH for 1 min and 30% EtOH for 1 min and water for 1 min
4. Treat with 0.06% KMnO4 dolution for 15 min (in dark for all processes from here)
5. wash with water for 1 min
6. stain with FluoroJade B solution (0.001% with added acetic acid drop) for 30 min
7. wash with water 1 min for 3 times
8. warm the sections in warmer
9. wash with xylene 2 min for 3 times
10 mount the sections
Now it is ready... Enjoy a lot I am doing with this quite successfully
see the below photograph of MCAo induced neuronal death of mice brain whrere infarct area (dead cells are in fluorescence)
Fluoro-jade
The staining solution was prepared from a 0.01% stock solution for Fluoro-Jade® B that was made by adding 10 mg of the dye powder to 100 mL of distilled water. To make up 100 mL of staining solution, 4 mL of the stock solution was added to 96 mL of 0.1% acetic acid vehicle. This results in a final dye concentration of 0.0004%. The stock solution, when stored in the refrigerator was stable for months, whereas the staining solution was typically prepared within 10 minutes of use and was not reused.
We have not done this lately, but we were successful when we did it.
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