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Tannic Acid (TA) Protocol

Stocks

  • Fixatives: e.g. [2.5%G/1%F or 0.1%G/4%F] in 0.1 M cacodylate + 3% sucrose + 1mM Mg3+
  • CB: cacodylate buffer, 0.1M pH 7.4
  • CB3S: cacodylate buffer, 0.1M pH 7.4, 3% sucrose
  • MB: maleate buffer, 0.1M pH 6.0
  • TA: 1% tannic acid in MB (sonicate & filter before use)
  • UAc: 1% uranyl acetate in MB (sonicate & filter before use)
  • PPD: 1% para-phenylenediamine in 75% ethanol

Procedure

  1. Fix with immersion or intraocular injection of desired fixative
  2. Follow with 30 m fixative on ice
  3. Store in 0.1M CB 3% sucrose if desired
  4. Rinse well 3 x 10 m in 0.1M CB on ice
  5. Incubate 40 m in TA ice
  6. Rinse well 2 x 10 m in MB on ice
  7. Incubate 40 m in dark UAc on ice
  8. Rinse well 2 x 10 m in MB on ice
  9. Wash 10 m in 75% ethanol on ice
  10. Incubate 15 m in PPD on ice (optional, enhances membranes but extracts Tannate)
  11. Rinse 3 x 10 m in 75% ethanol on ice (optional, enhances membrane but extracts Tannate)
  12. Rinse 10 m in 85% ethanol on ice
  13. Rinse 10 m in 95% ethanol on ice
  14. Rinse 2 x 10 m in 100% ethanol on ice
  15. Rinse 1 x 10 m in 100% acetone on ice
  16. Rinse 1 x 10 m in 100% acetone at RT
  17. 60 m 50% resin in acetone
  18. 4-20 h 75% resin in acetone
  19. 60 m 100% resin
  20. Embed samples in fresh resin in molds and cure at 60-65 deg C overnight

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