spermNMR methods #2: TUNEL Assay, part A: slide preparation

13 March 2020

This tutorial is a guide to preparing slides for the TUNEL assay. See the other Methods tutorial ‘TUNEL slide staining’ and ‘TUNEL analysis’ for further guides to the complete assay.

Required reagents and equipment

  • Polysine slides – polysine can only be used on one slide. This is the slide with the writing area on it

  • Hydrophobic barrier pen

  • Methanol


The ideal sperm concentration for counting is ~10 M/ml. The sample can be diluted with media, eg PBS, EBSS.


Create a paper template 22 x 22 mm (the size of a cover slip) to provide a guide for how much area to spread the sperm over.

Place the template under the polysine slide.

Step one

The technique for smearing sample onto the slide differs slightly for sperm and semen.


Pipette onto the middle of the slide. Using the edge of the pipette tip spread the sperm over the guide area (22x22mm) in a circular motion.


Preheat the polysine slide to 37°C to reduce the viscosity of semen when placed on the slide.

Using a positive displacement pipette add 10-15 ul of semen to the middle of the slide a smear using the full length of the pipette tip in a side to side motion.

Allow to dry for three hours.

Once dry, clean dried sample away from the edges of the slide to create a space for the wax border.

Step two

Border marking. Using the area template underneath the slide define the edges of the sample using a hydrophobic barrier pen.

Step three

Allow the wax to dry for five minutes.

Step four

Methanol fixing the sperm – This should be performed in a fume cupboard.

Step five

Using a Pasteur pipette add a drop of methanol over the entire sample within the wax border and wait one minute.

Step six

Tap off excess methanol.

Step seven

Dry for 30 minutes.

Step eight

Store slide in Dark slide box at -20°C.