Reduced silver staining

As used by Gabriella Wolff, Nicholas Strausfeld.

Bodian Protocol

1. Dissect tissue in cold AAF.

    a. 16 ml 80% Ethanol

    b. 1 ml Glacial AceticAcid

    c. 3 ml 25% Formaldehyde

2. Fix overnight at4°C.

3. The next morning, place dishes of paraffin in 60˚C oven to melt.

4. Dehydrate tissue inseries:

    a. 50% EtOH, 70% EtOH, 90% EtOH, 100% EtOH, 100% EtOH

        i. 10 minutes each

    b. Terpineol, xylenes, 60°C xylenes

        i. 10 minutes each

    c. Place tissue in 1:1 mixture of heated xylenes to paraffin for 15 min.

    d. Place tissue in 3 changes of paraffin for 20 minutes each.

5. Orient tissue in final paraffin dish and quickly place on an ice bath.

    a. Allow wax to cool overnight on ice bath.

6. Clean slides in 100% ethanol and allow to dry.

7. Coat slides with 1:4 mixture of Albumin fixative to distilled water.

    a. Slides must dry overnight in dust-free container.

8. The next day, trim paraffin and use hot spatula to transfer to cutting blocks.

    a. Wax should be trimmed in a trapezoid shape.

9. Section paraffin at 10-12 microns and float on distilled water on slides.

    a. Place slides on slide warmer to stretch sections.

    b. Use Ross Optical Lens Tissue to dry slide with rolling motion.

    c. Allow to dry overnight.

10. In the morning, pour 250 ml ddH₂O in a jar and place in 60°C oven.

11. Place slides in two changes of xylenes for 10 minutes each.

12. Rehydrate in series for 8 minutes in each step:

     a. Terpineol, 100% EtOH, 100% EtOH, 90% EtOH, 70% EtOH, 50% EtOH

     b. Final step in double distilled water (ddH₂O).

13. Right before end of hydration series, clean 2-6g copper filings (adjust according to species) with dilute nitric acid. e.g. For hermit crabs, use 6g copper.

     a. Copper can sit a short time in ddH₂O until ready to use.

14. Evenly distribute copper on the bottom of the heated jar of ddH₂O.

15. Sprinkle 2.5g Protargol* on the surface of the water and allow to sink in.

16. Incubate slides in Protargol solution overnight at 60°C.

17. In the morning, allow the jar to cool until room temperature.

18. Place slides in a jar of ddH₂O for 10-30seconds.

      a. It is important not to go over 30 seconds or staining is ruined.

19. Place slides in developer for 5 minutes

      a. 200 ml ddH₂O, 2g hydroquinone, 4g sodium sulfite.

20. Place in running tap water for 3 minutes.

      a. Do not direct water stream onto the slides and use a gentle current.

21. Place sections in 1% gold chloride under strong light for 10 minutes.

      a. Make sure gold chloride solution is filtered.

22. Place slides in two changes of ddH₂O for 30 seconds each.

23. Place slides in 200 ml ddH₂O with 4g oxalic acid for 8 minutes.

24. Place slides in two changes of ddH₂O for 30 seconds each.

25. Fix slides in 200 ml ddH₂O with 10g sodium thiosulfate for 5 minutes.

26. Wash slides in ddH₂O twice for 8 minutes each and dehydrate through xylenes.

      a. 50% EtOH, 70% EtOH, 90% EtOH, 100% EtOH, Terpineol, 2X xylenes.

27. Coverslip using Entellan mounting media.

*Note that Protargol is the Roques Proteinate d’Argent, which was discontinued around 1998-2000. The Strausfeld laboratory has no residual supply. “Silver proteinate” can be self-manufactured using a method described by: Pana, X., Bourlandb, W.A., Songa, W. (2013) Protargol Synthesis: An In-house Protocol. Journal of Eukaryotic Microbiology 60, 609–614.

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