Protocol for LR White Embedded Tissue for use w. TACS 2 TdT?
Posted: Mon 16 Feb 2015 15:24Do you have a supplementary protocol for LR White Embedded Tissue (Electron Microscopy) for use with the TACS® 2 TdT Core Kit?
FAQ from Trevigen
Protocol Guideline
1. Fix tissue in 4% paraformaldehyde, 0.2% glutaraldehyde, 2% sucrose, 0.1 M cacodylate buffer, pH 7.3.
2. Wash 3-4 times in 0.1 M cacodylate buffer, pH 7.3 (recipe below).
3. Incubate for 30 minutes in 0.1 M cacodylate buffer containing 1% sucrose.
4. Wash 4 times in 0.1 M cacodylate buffer.
5. Dehydrate in a graded ethanol series with 70% as the highest alcohol concentration.
6. Process LR White resin (G. Goping et al. (1999), J. Histochem. Cytochem. 44:289-295) followed by polymerization at 40ºC in vacuum oven.
7. Section at 90-95 nm and collect onto copper grids.
8. Etch sections for 10 seconds in 100% ethanol.
9. Rinse 3 times in PBS for 10 minutes each.
10. (In situ Apoptosis Detection: perform washes in porcelain dishes. For incubations, it is convenient to spot the reaction mixes onto Parafilm and float the grid section side down on the "drop".)
11. Immerse sample in 1X TdT Labeling Buffer (see Labeling Protocol) for 5 minutes.
12. Cover sample with 50 µl of Labeling Reaction Mix (see Labeling Protocol). Incubate for 60 minutes at 37°C in a humidity chamber.
13. Immerse sample in 1X TdT Stop Buffer (see Labeling Protocol) for 5 minutes.
14. Wash in dIH2O, 2 times for 2 minutes each.
15. Incubate sample in Streptavidin-Gold (e.g. at 2.5 µg/mL, 14-linker, from Eye Labs, Inc., San Mateo, CA) prepared in PBS + 1% BSA) overnight at 4ºC. Note that the concentration of Streptavidin-Gold and the incubation time will depend on source and some empirical testing will be needed.
16. Wash 4 times with PBS containing 1% BSA, 4 times in PBS, 3 times in dH2O and stain for 20 minutes in uranyl acetate and 0.4 minutes with lead citrate.
Note: Use highest quality water available (e.g. double distilled) and ensure blades used for sectioning are very clean for optimum results. Omit secondary fixation with osmium tetroxide (OSO4) and/or membrane enhancement with potassium ferricyanide of the tissue. DNA end-labeling on osmicated cells was 50% less efficient than labeling on unosmicated cells.
1 M Cacodylate Buffer:
* 300 mM Tris-Cl pH 7.3
* 1 M sodium cacodylate
* 10 mM cobalt chloride
NOTE: This protocol requires the separate purchase of Streptavidin-Gold conjugate.
HINTS: This kit has not been tested for use with resin embedded tissue. It should work with methacrylate.
FAQ from Trevigen
Protocol Guideline
1. Fix tissue in 4% paraformaldehyde, 0.2% glutaraldehyde, 2% sucrose, 0.1 M cacodylate buffer, pH 7.3.
2. Wash 3-4 times in 0.1 M cacodylate buffer, pH 7.3 (recipe below).
3. Incubate for 30 minutes in 0.1 M cacodylate buffer containing 1% sucrose.
4. Wash 4 times in 0.1 M cacodylate buffer.
5. Dehydrate in a graded ethanol series with 70% as the highest alcohol concentration.
6. Process LR White resin (G. Goping et al. (1999), J. Histochem. Cytochem. 44:289-295) followed by polymerization at 40ºC in vacuum oven.
7. Section at 90-95 nm and collect onto copper grids.
8. Etch sections for 10 seconds in 100% ethanol.
9. Rinse 3 times in PBS for 10 minutes each.
10. (In situ Apoptosis Detection: perform washes in porcelain dishes. For incubations, it is convenient to spot the reaction mixes onto Parafilm and float the grid section side down on the "drop".)
11. Immerse sample in 1X TdT Labeling Buffer (see Labeling Protocol) for 5 minutes.
12. Cover sample with 50 µl of Labeling Reaction Mix (see Labeling Protocol). Incubate for 60 minutes at 37°C in a humidity chamber.
13. Immerse sample in 1X TdT Stop Buffer (see Labeling Protocol) for 5 minutes.
14. Wash in dIH2O, 2 times for 2 minutes each.
15. Incubate sample in Streptavidin-Gold (e.g. at 2.5 µg/mL, 14-linker, from Eye Labs, Inc., San Mateo, CA) prepared in PBS + 1% BSA) overnight at 4ºC. Note that the concentration of Streptavidin-Gold and the incubation time will depend on source and some empirical testing will be needed.
16. Wash 4 times with PBS containing 1% BSA, 4 times in PBS, 3 times in dH2O and stain for 20 minutes in uranyl acetate and 0.4 minutes with lead citrate.
Note: Use highest quality water available (e.g. double distilled) and ensure blades used for sectioning are very clean for optimum results. Omit secondary fixation with osmium tetroxide (OSO4) and/or membrane enhancement with potassium ferricyanide of the tissue. DNA end-labeling on osmicated cells was 50% less efficient than labeling on unosmicated cells.
1 M Cacodylate Buffer:
* 300 mM Tris-Cl pH 7.3
* 1 M sodium cacodylate
* 10 mM cobalt chloride
NOTE: This protocol requires the separate purchase of Streptavidin-Gold conjugate.
HINTS: This kit has not been tested for use with resin embedded tissue. It should work with methacrylate.