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spermNMR methods #2: TUNEL Assay, part C: TUNEL analysis
13 March 2020
This tutorial is a guide to analysing stained washed sperm samples for the TUNEL assay. See the other Methods tutorial ‘TUNEL slide preparation’ and ‘TUNEL slide staining’ for further guides to the complete assay.
Required reagents and equipment
Inverted Fluorescent microscope
Immersion Oil
Method
Step one
Take slides from storage at 4°C in a dark slide box.
Step two
Place slide upside down on x20 lens to focus microscope.
Step three
The following illumination wavelengths are used to examine the fluorescent samples:
FITC – Ex/Em = 495/519 nm.
DAPI – Ex/Em = 345/455 nm.
Step four
Once sperm have been located, change to the x60 lens (remember add a drop of lens oil) and refocus at the higher magnification
Step five
Adjust the brightness level for x60 lens: FITC, ~85%; DAPI, 15-20%
Step six
Count at least 200 sperm from random locations of the slide, recording the numbers of DNA-fragmented sperm (green) and DNA-intact sperm (blue).
If no replicate slide, increase the number of sperm counted to 500.
