Sample type required: Deparaffinized and rehydrated tissue section (3–5 microns) on positively (+) charged slides
Preferred fixative: 10% neutral buffered formalin (NBF)
Control: Appendix, uterus
| Reagent | Time | Technical Notes |
| Bouin's solution | 15 minutes
(in heated solution) | Used as both a secondary fixative and mordant. Microwave solution for 1 minute before placing slides inside. Microwaved Bouin's solution should not be reused. |
| Running water wash | 5 minutes | Ensure that ALL picric acid (yellow) has been rinsed clear. |
| Weigert's working hematoxylin | 10 minutes |
|
| Running water wash | 5 minutes |
|
| Distilled water | 3 changes |
|
| Biebrich scarlet acid fuchsin | 5 minutes |
|
| Distilled water | 3 changes |
|
| Phosphotungstic/phosphomolybdic acid | 10 minutes | Discard the solution after use. DO NOT rinse slides after this step, as phosphotungstic/phosphomolybdic acid acts as a link between aniline blue stain and collagen. |
| Aniline blue | 5 minutes |
|
| Distilled water | 3 changes |
|
| <1% acetic acid | 1 minute | Discard the solution after use. |
Post staining procedure:
The tissue section should be dehydrated through graded alcohols (beginning with 95%). Two changes of xylene should follow, followed by a coverslip.
Expected results:
- Nuclei - Black
- Cytoplasm, muscle, erythrocytes - Red
- Collagen - Blue
