TITLE: Permanent specimen preparation by protargol staining
AUTHORS: Marek Valt, Michael Kotyk

[DESCRIPTION]
Protargol staining according to the protocol by Bodian (Bodian 1936) and later modified by Nie (Nie 1950), further optimized for staining of small flagellates.

[STEPS]
SECTION: Protargol staining optimized for a small flagellates, used for staining of Solarion arienae.
1. Mix 1 μm of culture sediment with a drop of sterile egg albumin, on a coverslip. 
For Solarion arienae, egg albumin was not diluted to achieve better adherence to the cells to coverslip.

SECTION: Protargol staining optimized for a small flagellates, used for staining of Solarion arienae.
2. Fix in Bouin-Hollande’s solution overnight. (0,175 mol/l picric acid, 0,138 mol/l Cu+2 acetate, 4% formaldehyde, and 5% acetic acid in water solution) 
SECTION: Protargol staining optimized for a small flagellates, used for staining of Solarion arienae.
3. Next day, transfer coverslips throught a graded ethanol series (70%, 50%).
SECTION: Protargol staining optimized for a small flagellates, used for staining of Solarion arienae.
4. Wash in distilled water.
SECTION: Protargol staining optimized for a small flagellates, used for staining of Solarion arienae.
5. Bleach in 0,5% KMnO4 solution for 5 minutes.
SECTION: Protargol staining optimized for a small flagellates, used for staining of Solarion arienae.
6. Wash in distilled water (5x).
SECTION: Protargol staining optimized for a small flagellates, used for staining of Solarion arienae.
7. Treat coverslips with 5% oxalic acid for 5 minutes.
SECTION: Protargol staining optimized for a small flagellates, used for staining of Solarion arienae.
8. Wash again in distilled water (5x).
SECTION: Protargol staining optimized for a small flagellates, used for staining of Solarion arienae.
9. Stain in 1% protargol solution, for 48 hours at 37°C in a beaker with copper wire pieces placed between the coverslips.

For Solarion, protargol produced by Bayer, I. G. Farbenindustrie Actinengesellschaft (out of business since 1952) was used. 
SECTION: Protargol staining optimized for a small flagellates, used for staining of Solarion arienae.
10. Wash in distilled water (2x).
SECTION: Protargol staining optimized for a small flagellates, used for staining of Solarion arienae.
11. Treat with freshly prepared reducing solution (1% hydroquinone and 5% Na2SO3), for 10 minutes.

SECTION: Protargol staining optimized for a small flagellates, used for staining of Solarion arienae.
12. Wash in distilled water (5x).
SECTION: Protargol staining optimized for a small flagellates, used for staining of Solarion arienae.
13. Tone with 1% AuCl3, for 5 minutes. 
SECTION: Protargol staining optimized for a small flagellates, used for staining of Solarion arienae.
14. Wash in distilled water (2x).
SECTION: Protargol staining optimized for a small flagellates, used for staining of Solarion arienae.
15. Treat with 2% oxalic acid, for 5 minutes.
SECTION: Protargol staining optimized for a small flagellates, used for staining of Solarion arienae.
16. Wash in distilled water (5x).
SECTION: Protargol staining optimized for a small flagellates, used for staining of Solarion arienae.
17. Treat with 5% Na2S2O3 for 10 minutes
SECTION: Protargol staining optimized for a small flagellates, used for staining of Solarion arienae.
18. The final wash is done under a constant stream of tap water for 20 minutes. 
SECTION: Protargol staining optimized for a small flagellates, used for staining of Solarion arienae.
19. Dehydrate the coverslips in an ethanol series (50%, 70%, 80%, 96%, 100%)
and 3 times in xylene (5 minutes each). 
SECTION: Protargol staining optimized for a small flagellates, used for staining of Solarion arienae.
21. Finally, the stained, dehydrated coverslips are mounted on glass slides with DPX mounting
medium (Sigma–Aldrich).
SECTION: Protargol staining optimized for a small flagellates, used for staining of Solarion arienae.
20. Dehydrate in xylene, 3 times (5 minutes each).