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 solution after use. DO NOT rinse slides after this step as phosphotungstic/phosphomolybdic acid acts as a link for aniline blue stain to collagen. |
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 though graded alcohols (beginning with 95%). Follow with two changes of xylene and then coverslip.
Expected results:
- Nuclei - Black
- Cytoplasm, muscle, erythrocytes - Red
- Collagen - Blue