This procedure is performed after polar body removal has been performed. Polar body cells are biopsied from unfertilized and fertilized oocytes by mechanical dissection.
- Prepare fixative (methanol:glacial acetic acid, 3:1) and store in freezer until ready to use.
- Prepare coplin jar containing methanol.
- Engrave a small circle in the bottom of a 35 x 10 mm petri dish. The circle identifies the target area in which to place the polar body allowing for easier relocation.
- Write the specimen identification and pertinent information on the frosted area of the glass microscope slide(s). Pre-clean the slide by wiping with fixative and a lint-free cloth.
- Fill a hanging drop slide with purified water.
- Heat the tip of a 25 µL capillary pipette using a microtorch and simultaneously pull on the heated pipette tip to extend and create a tip with a 50 µm inner diameter. Insure that the extended tip of the pipette has an even break on is so it is not sharp. A sharp tip may damage the polar body. Break the tip again, if necessary in order to create a smooth tip. The size of the pipette opening should be checked prior to picking up the polar body to ensure that it is the proper size; if the diameter is too large, there is a greater potential for loss of the polar body.
- Aspirate a small amount (approximately 2 to 3 µL) of water into the pipette.
- Using a stereomicroscope locate the polar body in the petri dish.
- Gently aspirate the polar body into the pipette and transfer it to the hanging drop slide containing the water. Release the polar body from the pipette by gently blowing, placing it within the center circle of the hanging drop slide.
- Move the polar body from the water by aspirating it along with a small amount of water into the attenuated pipette. Transfer the cell to a microscope slide with a small amount of water. Inscribe a circle on top of the slide around the drop of water with a carbide marker.
- Allow the polar body to dry in the drop of water on the slide by constantly viewing under the inverted microscope. When dry, aspirate asmall amount of fixative (approximately 2 to 3 µL) into the same attenuated pipette and drop the fixative onto the polar body.
Note: When fixing first polar bodies 6 hours from the time of retrieval, drip fixative just prior to complete drying in order to remove all cytoplasm.
- Place another drop of fixative from the attenuated pipette over the cell before complete drying unless dissolution of the cytoplasm is obvious and has occurred. Repeat until the cytoplasm surrounding the cell has dissolved leaving only the nucleus (apply fixative at least 5 times).
Note: Complete removal of the cytoplasm is essential to optimum probe hybridization.
- Using the scribe, draw two circles around the chromatin on the top side of the glass slide. One circle is drawn slightly larger than the other circle, i.e., one circle is inside the other with the cell in the middle of the smaller circle. The circle inscribed around the cell will allow for easier cell location after the hybridization procedure.
- Place the slide in the coplin jar containing methanol for 5 minutes.
- Remove the slide from the methanol and allow to air dry.
- Using a vernier scale on the microscope stage, or an England Finder, locate and record the cell location on the microscope slide. These coordinates will help in the location of the cell after hybridization.
- Draw another small circle on the bottom of the microscope slide around the cell using a permanent marker. This defines the area in which to apply the probe.