Thionin Staining Protocol as of 2/5/13
Protocol based on the 2003 procedure (on the wall in the wet lab)
Part 1: de-lipidation
1. 70% ethyl alcohol (EtOH) 2 changes, 2 min each
2. 95% EtOH 2 solutions, 2 min each
3. 100% EtOH 4 solutions, 2 min each
4. Xylene 3 solutions, 15 min each (under hood)
Part 2: rehydrating
1. 100% EtOH 4 solutions, 2 min each
2. 95% EtOH 2 solutions, 2 min each
3. 70% EtOH 2 solutions, 2 min each
4. 50% EtOH 1 solution, 2 min
5. deionized H2O (dH2O) 1 solution, 2 min
Part 3: staining and differentiation
1. dip in thionin dye, 30 seconds
2. dip in dH2O, 30 seconds
3. 50% EtOH 1 solution, 2 min
4. 70% EtOH 2 solutions, 2 min each
5. 95% EtOH 2 solutions, 2 min each
6. If overstained, differentiate in 95% EtOH with glacial Acetic Acid (1.5 ml gAA/250 ml EtOH)*
7. 100% EtOH 4 solutions, 2 min each
8. Xylene 3 solutions, 5 min each
9. Coverslip and dry flat
Jaspar that's great. Exactly the format I had in mind, and would definitely work, but I think we can streamline it a little more. Now that we have a protocol we can note differences in what we actually do and then update and annotate published protocol (or a copy of it) afterward.
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