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spermNMR methods #2: TUNEL Assay, part C: TUNEL analysis

Posted on March 13th 2020 in Blog, Methods, News
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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/equipment:

Inverted Fluorescent microscope

Immersion Oil


1. Take slides from storage at 4°C in a dark slide box.

2. Place slide upside down on x20 lens to focus microscope.

3. The following illumination wavelengths are used to examine the fluorescent samples:

FITC – Ex/Em = 495/519 nm.

and

DAPI – Ex/Em = 345/455 nm.

4. Once sperm have been located, change to the x60 lens (remember add a drop of lens oil) and refocus at the higher magnification

5.  Adjust the brightness  level  for x60 lens: FITC, ~85%; DAPI, 15-20%

6. 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

 

Fluorescent visualization at 400x magnification of DNA-fragmented sperm (green) and DNA-intact sperm (blue, DAPI)