spermNMR methods #2: TUNEL Assay, part C: TUNEL analysis
13 March 2020
Required reagents and equipment
Inverted Fluorescent microscope
Take slides from storage at 4°C in a dark slide box.
Place slide upside down on x20 lens to focus microscope.
The following illumination wavelengths are used to examine the fluorescent samples:
FITC – Ex/Em = 495/519 nm.
DAPI – Ex/Em = 345/455 nm.
Once sperm have been located, change to the x60 lens (remember add a drop of lens oil) and refocus at the higher magnification
Adjust the brightness level for x60 lens: FITC, ~85%; DAPI, 15-20%
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.