TUNEL Assay Kit - HRP-DAB

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This is a protocol summary; please use the downloadable protocol booklet for full instructions.

TUNEL assay protocol for HRP-DAB TUNEL staining

Use for paraffin-embedded tissue sections, frozen tissue sections, or cell preparations.

Notes

- to contain small reagent volumes, encircle specimens using a hydrophobic slide marker
- after most protocol steps carefully remove excess liquid and dry around the specimen
- pulse-spin reagents in a microcentrifuge prior to opening
- during TdT enzyme labelling, ensure temperature is >22 ºC and use a humidified chamber (for example place slides on moist paper towel and then cover)
- warm the Stop Buffer to 37ºC before use to remove precipitate
- keep enzymes and enzyme reagent mixes on ice
- 100 μL of solution is generally enough to cover a specimen on a slide
- always prepare fresh DAB working solution from the 2 reagents, just before use
- to avoid loss of sample from glass slides during washing steps, dip slides 2-3 times into TBS instead of rinsing with a wash bottle
- do not let the specimen dry out during or between any steps. If necessary, cover or immerse the specimen in TBS.

Paraffin embedded tissue sections

Rehydrate slides in xylene, and decreasing ethanol (100%,90%,80%,70%), and then TBS.

Permeabilize tissue by incubation for 20mins with Proteinase K, then wash with TBS. (Incubate frozen sections for 10mins and cell preps for 5mins.)

Quench and inactivate endogenous peroxidases by incubation in 3% H2O2 in methanol for 5 min, then wash with TBS.

Equilibrate with TdT Equilibration Buffer for 30 minutes.

For labelling, incubate with TdT enzyme mix for 1.5 hours, using a coverslip to prevent drying.

To terminate labeling, submerge the slide in TBS and allow the coverslip to slide off, wash with TBS, incubate with stop buffer for 5 min, and wash again with TBS.

Block with Blocking Buffer for 10 min, and then incubate with Conjugate in blocking buffer for 30 min (use a humidified chamber), then wash with TBS.

Incubate in DAB working solution for 15 min, counterstain with Methyl Green solution for 1-3 min.

Finally dehydrate slides in 100% ethanol and xylene, and mount with organic mounting media.

Frozen tissue sections and cell preparations

Fix slides with frozen tissue sections in 4% formaldehyde in PBS for 15 minutes, and wash in TBS.

Fix cell preparations in 4% formaldehyde in PBS at 10^6 cells/mL for 10 minutes. Then pellet cells and re-suspend in 80% ethanol. Air-dry cells onto glass slides or use a cyto-spin. Rehydrate in TBS for 15 mins.

Then follow protocol for paraffin embedded sections.

Analysis

Apoptosis is represented by a dark brown (DAB) signal in light microscopy. Lighter shades of brown and/or shades of blue-green to green—brown indicate a nonreactive/negative cell.

For a positive control, fragment DNA in a specimen by generating free 3’OH groups identical to those generated during apoptosis, through treatment with DNase I.

For a negative control, omit the TdT enzyme in the reaction mix.