150 Connective Tissue (chap. 13) 



Phosphotungstic acid: 



phosphotungstic acid 5.0 gm. 



distilled water 100.0 ml. 



Aniline blue stain: 



aniline blue, WS, C.I. 42780 0.5 gm. 



orange G, C.I. 16230 2.0 gm. 



oxalic acid 2.0 gm. 



distilled water 100.0 ml. 



5% phosphotungstic acid (above) 1.0 ml. 



Acidulated water: 



glacial acetic acid 1.0 ml, 



distilled water 100.0 ml. 



procedure: 



1. Deparaffinize and hydrate slides down to water; remove HgCU. 



2. (aniline alcohol: 45 minutes)^ 



3. (acid alcohol: 1-2 minutes) 



4. Stain in azocarmine, 56°C: 1 hour. (2 hours) Check temperature 

 carefully; if too high, azocarmine differentiates poorly. 



5. Rinse in distilled water. 



6. Differentiate in aniline alcohol. Check inider microscope for 

 brilliant red nuclei and very light red cytoplasm. 



7. Treat with acid alcohol: 1-2 minutes. 



8. Transfer to phosphotungstic acid: 2-3 hours. (4 hours) 



9. Rinse in distilled water. 



10. Stain in aniline blue sokuion: 1-2 hours. (4 hours) 



1 1 . Rinse in distilled water. 



12. Treat with phosphotungstic acid: 3-5 minutes. 



13. Rinse in distilled water. 



14. Rinse in acidulated water: 1-2 minutes or lonoer. 



15. Rinse in 70% alcohol: briefly dip. 



16. Dehydrate 95% alcohol, several dips in each of 2 changes. 



17. Dehydrate, absolute alcohol, clear and mount. 



results: 



nuclei — brilliant red 

 collagen and reticulum — blue 

 muscle — red and yellow 

 basophil cytoplasm — light bkie 



^ For strikingly beautiful results, particularly for pituilarv . Konelf's longer niethod is 

 recommended. Changes in timing are indicated in parentheses. 



