This is a powerful stain for tissue that has been fixed too long in Bouin's (over 2 days) or in formalin (over 6 months). Standard thionin formulations will not stain over-fixed tissues.

The trick appears to be use of the aniline in the stain solution.

1 ml Aniline, fresh

10 ml Absolute ethanol

0.25 gm Thionin

Deionized water

1. Add 1 ml of aniline to 10 ml of absolute ethanol.
2. Shake well;
3. Bring up to 100 ml with deionized water;
4. Add 0.25 gm thionin and stir well;
5. Filter into a bottle and keep well stoppered.

Used stain can be filtered back into the bottle after each staining. Stain is good for years on the shelf.

1. Deparaffinize and hydrate tissue, as usual;
2. Stain for 10 min. in Putt's Thionin;
3. Rinse in 2 changes of deionized water;
4. Begin differentiation in 95% EtOH for about 5 min.;
5. Place tissue in second 95% EtOH to which has been added 1 drop of glacial acetic acid per 300 ml of EtOH;

Note: you may need 2 or 3 drops per 100 ml EtOH for normal tissue, or to stain for less time.

6. Move to 100% when tissue is almost differentiated, or at least 30 sec;
7. Clear in Cajeput oil for 5 min.;
8. Clear through 3 or 4 changes of xylene;
9. Coverslip.

From old Rubelab procedure manual.