spermNMR methods #2: TUNEL Assay, part B: slide staining

13 March 2020

This tutorial is a guide to staining washed sperm samples for the TUNEL assay. See the other Methods tutorial ‘TUNEL slide preparation’ and ‘TUNEL analysis’ for further guides to the complete assay.

Required reagents

Room temperature

  • 10mM Tris, pH 8

  • x1 Tris buffered saline (TBS), pH 7.6

  • x1 PBS, pH 7.4

  • DAPI (mounting media

On ice

  • Proteinase K, 2mg/ml (PK)

  • TdT Equilibration buffer, x5 (EB)

  • TdT Labelling Reaction Mix (RM)

Warning, do not let the specimen dry out during or between any steps.

(if necessary, cover or immerse the specimen in 1X TBS to keep hydrated)

In order to maintain protocol timing it is recommend staining up to 12 slides at any one time.


Step one

Remove slides from the freezer and place on a staining tray.

Step two

Wash sperm by placing a drop of ~1ml, x1 TBS on each slide for 15 minutes at room temperature (Rehydration step).

Warning: the wax border can detach during washing.

If this occurs tap off excess liquid (ensuring that the sperm do not dry out). Dry the area where the wax has come away and re-apply a new border.

Solution A: Prepare 20 mg/ml PK (Permeabilization step); keep on ice until used.

Step three

Tap off excess liquid from each slide and use a paper towel in the corner of the wax border to remove additional fluid.

Step four

Add 100 µl of Solution A and incubate for five minutes at room temperature.

Solution B: Prepare x1 EB on ice (Equilibration step); keep on ice until used.

Step five

Wash slide with 1 ml x1 TBS 3 times, tapping off liquid between each wash as step three.

Step six

Add 100 µl of Solution B.

Step seven

Incubate for 30 minutes at room temperature (cover the staining tray with its lid during incubation).

Step eight

Remove TdT enzyme from freezer shortly before the next step.

Negative control: replace TdT enzyme with dH2O

Solution C: Prepare RM (Labelling reaction step). On ICE!

Step nine

Tap excess liquid off each slide, as step three.

Step ten

Add 50 – 60 µl of Solution C.

Step 11

Cover each slide with Parafilm. To prevent sample leaking ensure that no fluid is outside of the wax border. Do not press down on the Parafilm.

Step 12

Place each slide in a pre-warmed (37°C), tin foil wrapped ’Humidifier chamber’ containing damp paper towel (the slides require light protection).

Step 13

Place chamber in an incubator for 60 – 90 minutes at 37°C.

Step 14

Remove Parafilm and wash slide three times with 1 ml x1 PBS. Tap off excess liquid (step three) and wait ~1 minute between each wash.

Step 15

Add 100 µL of DAPI counter stain to each slide and place a coverslip on top of each sample. Seal the coverslip with clear nail varnish.

Step 16

Store at 4°C in dark slide box.

Next, go to Methods: TUNEL analysis