spermNMR methods #2: TUNEL Assay, part B: slide staining
13 March 2020
10mM Tris, pH 8
x1 Tris buffered saline (TBS), pH 7.6
x1 PBS, pH 7.4
DAPI (mounting media
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.
Remove slides from the freezer and place on a staining tray.
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.
Tap off excess liquid from each slide and use a paper towel in the corner of the wax border to remove additional fluid.
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.
Wash slide with 1 ml x1 TBS 3 times, tapping off liquid between each wash as step three.
Add 100 µl of Solution B.
Incubate for 30 minutes at room temperature (cover the staining tray with its lid during incubation).
Remove TdT enzyme from freezer shortly before the next step.
Solution C: Prepare RM (Labelling reaction step). On ICE!
Tap excess liquid off each slide, as step three.
Add 50 – 60 µl of Solution C.
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.
Place each slide in a pre-warmed (37°C), tin foil wrapped ’Humidifier chamber’ containing damp paper towel (the slides require light protection).
Place chamber in an incubator for 60 – 90 minutes at 37°C.
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.
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.
Store at 4°C in dark slide box.
Next, go to Methods: TUNEL analysis