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