Sample type required: Deparaffinized and rehydrated tissue section (3-5 microns) on positively (+) charged slides
Preferred fixative: 10% neutral buffered formalin (NBF)
Control: Internal; appendix, fallopian tube, uterus, or small intestine are recommended choices
Bouin's solution | 15 minutes (in heated solution) | Used as both a fixative and mordant. Microwave solution for 1 minute before placing slides inside. |
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 | Required for nuclear bluing. |
Distilled water | 3 changes |
|
Biebrich scarlet acid fuchsin
| 5 minutes |
|
Distilled water | 3 changes |
|
Phosphotungstic/phosphomolybdic acid
| 10 minutes | Discard solution after use. |
Aniline blue | 5 minutes |
|
Distilled water | 3 changes |
|
< 1% acetic acid
| 1 minute | Discard solution after use. |
Post staining procedure: Tissue section should be dehydrated with 95% and absolute alcohols. Follow with two changes of xylene and then coverslip.
Expected results:
- Nuclei - Black
- Cytoplasm, muscle, erythrocytes - Red
- Collagen - Blue