spermNMR methods #2: TUNEL Assay, part A: slide preparation:
Posted on March 13th 2020 in Blog, Methods, News
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.
Polysine slides – polysine can only be used on one slide. This is the slide with the writing area on it
Hydrophobic barrier pen
The ideal sperm concentration for counting is ~10 M/ml. The sample can be diluted with media, e.g. 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
1. 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 3 hours
Once dry, clean dried sample away from the edges of the slide to create a space for the wax border.
2. Border marking. Using the area template underneath the slide define the edges of the sample using a hydrophobic barrier pen.
3. Allow the wax to dry for 5 minutes
4. Methanol fixing the sperm – This should be performed in a fume cupboard
5. Using a Pasteur pipette add a drop of methanol over the entire sample within the wax border and wait one minute
6. Tap of excess methanol
7. Dry for 30 minutes
8. Store slide in Dark slide box and -20°C