Phase Contrast Microscopy

1. Testis buffer: 183 mM KCl, 47 mMNaCl, 10 mMTris-HCl, pH 6.8 or TBI: 15 mM potassium phosphate (equimolar dibasic and monobasic), pH 6.7, 80 mM KCl, 16 mM NaCl, 5 mM MgCl2, 1% polyethylene glycol (PEG) 6000.

Fig. 5. (opposite page) Meiosis I progression in wild-type spermatocytes observed by time-lapse phase-contrast and fluorescence microscopy. Meiosis was followed from early prometaphase to the end of meiosis I at a rate of 20 frames/min using a combination of phase contrast (left panels) and fluorescence (right panels) microscopy. The chromosomes were labeled with a His2-GFP fusion protein (right panels). Five significant time-points are shown in this figure, reproduced from Rebollo and Gonzalez (41): 0' corresponds to late prophase, the two centrosomes have migrated to opposite poles and organized large asters. The meiotic spindle is forming and is highlighted with phase dark mitochondria (asterisks). A nonchromosomal phase dark nuclear structure, which remains throughout meiosis, can be seen (arrowhead). At 32', the sperma-tocyte contains a fully formed elongated spindle. Two bivalents are stabilized at the metaphase plate in this focal plane (arrows). At 37', anaphase has just started. Two pairs of homologous chromosomes can be seen segregating from each other (double arrows). At 41', the chromosomes have reached the poles and are decondensing. Chromosome decondensation is first apparent midway through anaphase. At 55', the two daughter nuclei have formed (arrows) and the cleavage furrow begins to pinch the central spindle (white arrowheads).

Drosophila Accessory Glands

Fig. 6. Other cell types and typical artefacts that should be ignored. (A) Cells from the accessory gland can be confused with early primary spermatocytes. However, primary spermatocytes are in cysts, the nuclei of accessory gland cells are more uniform, and there is never a polarity to the accessory gland cytoplasm. (B) Oversquashed primary spermatocytes can develop vacuoles adjacent to the nucleus. These can be confused with mutant onion stages; however, the large spermatocyte nuclei are usually still apparent. (C) The cytoplasmic waste bag extruded upon individualization can sometimes be confused with a very disorganized early elongation cyst. (D) Cells can fuse under pressure from the cover slip. Fused normal onion stage cells can be confused for cytokinesis mutants. True cytokinesis mutants show two or four phase light nuclei adjacent to only one (large) Nebenkern. Here, the nuclei and Nebenkern are similar in size and equal in number. (E) Occasionally, when elongated cysts are disrupted just before individualization the sperm tails have a blebby rather than smooth appearance. For this to be classified as the mutant phenotype, it has to be consistently rather than rarely seen. (Scale bar is 10 |im and applies to all panels, except panel C.)

Fig. 6. Other cell types and typical artefacts that should be ignored. (A) Cells from the accessory gland can be confused with early primary spermatocytes. However, primary spermatocytes are in cysts, the nuclei of accessory gland cells are more uniform, and there is never a polarity to the accessory gland cytoplasm. (B) Oversquashed primary spermatocytes can develop vacuoles adjacent to the nucleus. These can be confused with mutant onion stages; however, the large spermatocyte nuclei are usually still apparent. (C) The cytoplasmic waste bag extruded upon individualization can sometimes be confused with a very disorganized early elongation cyst. (D) Cells can fuse under pressure from the cover slip. Fused normal onion stage cells can be confused for cytokinesis mutants. True cytokinesis mutants show two or four phase light nuclei adjacent to only one (large) Nebenkern. Here, the nuclei and Nebenkern are similar in size and equal in number. (E) Occasionally, when elongated cysts are disrupted just before individualization the sperm tails have a blebby rather than smooth appearance. For this to be classified as the mutant phenotype, it has to be consistently rather than rarely seen. (Scale bar is 10 |im and applies to all panels, except panel C.)

2. Dissecting plate; for example, a 10-cm-diameter plastic Petri dish with the sides removed.

3. Sharp forceps (the sharper the better).

4. Tungsten mounted needles (sharp!).

5. Microscope slides and cover slips, 22 x 22 mm2.

6. Phase-contrast compound microscope with camera.

7. Kimwipes.

8. Hoechst 33342 (bis-benzimide; e.g., Sigma-Aldrich) (1 mg/mL stock). Store in the dark at 4°C. Dilute in dissection buffer to 2-5 |g/mL when needed.

10. Halocarbon oil (Voltalef 10S).

Was this article helpful?

0 0

Post a comment