Acridine Orange Staining

Acridine orange is a vital dye that differentially stains living and dying cells (Fig. 2). The advantage of acridine orange is that it is performed quickly on live tissue. However, this also means that the tissue must be examined and photographed immediately after staining. These protocols are derived from protocols described in refs. 15 and 43.

3.3.1. Embryo Protocol

1. Dilute AO stock solution to 5 ^g/mL in 0.1 M phosphate buffer.

2. Collect embryos in mesh baskets as described and wash only in water (see Note 7). Using a fine-tipped paintbrush, transfer embryos from the mesh to tubes containing an equal volume of heptane and the 5 |Jg/mL AO solution. Microcentrifuge tubes or glass tubes with tight-fitting caps may be used.

3. Shake tubes vigorously by hand for 3-5 min. Shaking by hand improves the permeability of the embryos (see Note 8).

4. Pipet off the liquid and replace with heptane.

5. Pipet the embryos in heptane onto glass slides. Try to keep the embryos separated and soak up the heptane using a Kimwipe twisted into a point (see Note 9). Quickly cover the embryos with halocarbon oil and a cover slip.

6. View the slide immediately under epifluorescence. AO staining is visible under both rhodamine and fluorescein filters. The rhodamine filter often looks

Fig. 3. Annexin V labeling of egg chambers. (A) Intense staining is seen on nurse cell membranes at stage 10, but not in the smaller stage 8 egg chamber. (B) Higher magnification (400x) reveals punctate staining on the membrane (reduced from original magnification).

better, as the fluorescein filter shows more background and smearing from residual heptane.

3.3.2. Ovary Protocol

1. Dilute AO stock solution to 10 | g/mL in phosphate buffer.

2. Transfer dissected ovaries to an Eppendorf tube containing 15 |L of heptane and 15 |L of 10-|g/mL AO solution.

3. Flick the tube gently to mix and allow to rotate for 5 min.

4. Transfer ovaries to slides and spread out the ovary tissue into individual egg chambers if possible. Pipet off the AO/heptane mixture or use a Kimwipe twisted into a point (see Note 9). Cover with halocarbon oil and a cover slip.

5. View the slide immediately under epifluorescence, using the fluorescein, rhodamine, or UV filter. Under UV, the apoptotic nuclei stain yellow or red (43).

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