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spermNMR methods #3: Capacitation Status: Tyrosine phosphorylation staining
4 January 2022
This tutorial is a guide to preparing, staining and analysing slides for tyrosine phosphorylation staining to determine the percentage of capacitated sperm.
Part A: slide preparation
Required reagents and equipment
Polysine slides (can only be used on the side with the writing area on it)
Hydrophobic barrier pen
Methanol
The ideal sperm concentration for counting ~10 M/ml. The sample can be diluted with media, e.g. PBS, EBSS.
Protocol
Create a template with two 22 x 22 mm (the size of a cover slip) square cut outs to provide a guide for how much area to spread the sperm over.
Step one
Pipette two sample spots onto the slide. Using the edge of the pipette tip, spread the sperm over each of the guide areas (22x22mm) in a circular motion.
Step two
Allow to dry for three hours at room temperature (can leave maximum of 24 hours).
Step three
Once dry, clean dried sample away from the edges of the slide to create a space for a wax border.
Step four
Border marking. Using the area template underneath the slide define the edges of the sample using a hydrophobic pen.
Step five
Allow the wax to dry for five minutes.
Step six
Methanol fixing the sperm – This should be performed in a fume cupboard.
Step seven
Using a Pasteur pipette, add a drop of methanol over the entire sample within the wax border and wait one minute.
Step eight
Tap off excess methanol.
Step nine
Dry for 30 minutes in the fume cupboard.
Step ten
Store slide in dark slide box at -20°C.
Part B: slide staining
Required reagents and equipment
1 x TBS (note: PBS is not suitable for washing as it interferes with phosphorylated tyrosine residues)
Antibody diluent
Primary ?-phosphotyrosine mouse monoclonal antibody (clone 4G10)
Secondary antibody – rabbit-?-mouse IgG-fluorescein isothiocyanate conjugated
Pro-long antifade mounting media
Protocol
Step one
Remove slides from the freezer and place on a staining tray. In order to maintain protocol timing it is recommend staining up to 10 slides at any one time.
Step two
Wash sperm by placing a drop of ~1ml, x1 TBS on each sperm smear for 15 minutes at room temperature.
Step three
Tap off excess liquid from each slide and use a paper towel in the corner of the wax border to remove additional fluid.
Step four
Add 100 ?l of primary antibody, diluted 1:500 in antibody diluent (can be prepared during 15 minutes incubation in TBS), to each smear and place each slide in a pre-warmed (37°C), tin foil wrapped ’Humidifier chamber’ containing damp paper towel.
Step five
Incubate for 60 minutes at 37°C.
Step six
On each slide, wash the sperm smears with 1 ml x1 TBS, repeating three times, tapping off the liquid between each wash as step three, and wait ~ one minute between each wash.
Step seven
Add 100 ?l of secondary antibody (diluted 1:100 in antibody diluent) per smear and place each slide back in the pre-warmed (37°C), tin foil wrapped ’Humidifier chamber’ containing damp paper towel.
Step eight
Incubate for 60 minutes at 37°C.
Step nine
Again, wash each smear with 1 ml x1 TBS three times, tapping off the liquid between each wash as step three, and wait ~ one minute between each wash.
Step ten
Add a drop of pro-long antifade mounting media to each slide smear and place a cover slip on top of each smear. It is recommended to use a dark coloured staining tray to help identify any bubbles – if any are present lightly press with a pipette tip to remove.
Step 11
Seal the coverslip with clear nail varnish and allow to dry for approximately ten minutes.
Step 12
Store at 4°C in dark slide box until analysis.
Part C: slide analysis
Required reagents and equipment
Inverted fluorescent microscope
Immersion oil
Protocol
Step one
Take slides from storage at 4°C in dark slide box.
Step two
Place slide upside down on x20 lens to focus microscope.
Step three
The following parameters are used to examine the fluorescent samples:
FITC – Ex/Em = 495/519nm
Step four
Once cells have been identified, add a drop of lens oil to the x60 lens and place slide upside down.
Step five
Count at least 200 sperm from random locations of the sperm smear, recording the different staining patterns. If no replicate smear on the slide, increase the number of sperm counted to 500.
