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spermNMR methods #2: TUNEL Assay, part A: slide preparation
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
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
Method
Method
The ideal sperm concentration for counting is ~10 M/ml. The sample can be diluted with media, eg PBS, EBSS.
Preparation
Preparation
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
Step one
The technique for smearing sample onto the slide differs slightly for sperm and semen.
Sperm
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.
Semen
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
Step two
Border marking. Using the area template underneath the slide define the edges of the sample using a hydrophobic barrier pen.
Step three
Step three
Allow the wax to dry for five minutes.
Step four
Step four
Methanol fixing the sperm – This should be performed in a fume cupboard.
Step five
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
Step six
Tap off excess methanol.
Step seven
Step seven
Dry for 30 minutes.
Step eight
Step eight
Store slide in Dark slide box at -20°C.
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