- Make Sybr 14 working solution:
a. Add 20 µl Sybr 14 (from Invitrogen kit) to 980 µl DMSO
b. Store at -20°C in the dark.
- Make sufficient volume of sperm diluent of HEPES, NaCl, and BSA (HNB). See Table 4
- For spermathecae
a. Transfer diluent with sperm (from step 5.4.1.) to a labelled 2 ml glass vial.
b. After all spermathecae have been dissected, add 10 µl Sybr 14 in DMSO to each vial.
c. Incubate 5-10 min at 36°C.
d. Add 5 µl propidium iodide.
e. Incubate 5-10 min at 36°C.
- For seminal vesicles
Gently disrupt seminal membrane to release sperm in diluent.
a. Discard seminal membrane and pour sperm solution into a 2 ml glass vial.
b. Stain seminal vesicles:
i. Add 10 µl Sybr 14/DMSO.
ii. Incubate 15 min at 36°C.
iii. Add 6 µl propidium iodide.
iv. Incubate another 15 min at 36°C.
- Turn on fluorescent lamp and camera, and open photo software on computer.
- Use microdispenser to load haemocytometer with 10 µl sperm solution across both chambers.
- Place haemocytometer on microscope table and examine under low magnification to centre view on grid (Fig. 26).
- Switch to high magnification (200x then 400x).
- Turn off visible light and open fluorescence. Focus using FITC filter, then take an image using the camera. This will be a picture of the dead sperm in the field.
- Without moving the haemocycometer, switch to Rhodamine filter and take another image. This will be a picture of the live sperm in the same field.
- Move to a new field of view and repeat; a total of five replicates should be minimum, preferably greater particularly if there is large variation among fields.
- Save pictures using descriptive names indicating sample number, live or dead sperm, and picture number.
- Switch back to low-resolution and close fluorescent lamp aperture.
- Clean haemocytometer and cover slip with a kimwipe.
- Repeat for each new sample.