Dernburg Abby F
Cold Spring Harb Protoc. 2011 Dec 1;2011(12):1531-3. doi: 10.1101/pdb.prot066894.
This article describes a procedure for the manual dissection and fixation of Drosophila egg chambers for whole-mount fluorescent in situ hybridization (FISH). Egg chambers are more challenging to fix than embryos. They are easily overfixed so that probes will not penetrate well into the oocyte nucleus, even if some hybridization is detected in the follicle and nurse cells closer to the surface. If late-stage oocytes are to be examined, the tissue must also be protected against osmotic shock before and during fixation, because this will activate metaphase-arrested nuclei to undergo the metaphase-anaphase transition. Warming the buffered formaldehyde to 30°C immediately prior to fixation tends to enhance probe penetration. In contrast to reported results with immunostaining, FISH is equally successful whether fixation is performed with freshly prepared formaldehyde or commercial formaldehyde solutions. Manual dissection of fixed ovarioles gives a much higher yield per adult than the blender technique (facilitating analysis of mutants), and younger egg chambers in particular will be enriched, although the procedure itself is more laborious.
本文介绍了一种用于手动解剖和固定果蝇卵室以进行全片荧光原位杂交（FISH）的方法。卵室比胚胎更难固定。它们很容易过度固定，以至于即使在靠近表面的卵泡和滋养细胞中检测到一些杂交信号，探针也无法很好地穿透到卵母细胞核中。如果要检查晚期卵母细胞，在固定前和固定过程中还必须保护组织免受渗透压休克的影响，因为这会激活中期停滞的细胞核进入中期-后期转变。在固定前立即将缓冲甲醛加热至30°C往往会增强探针的穿透能力。与免疫染色的报道结果相反，无论使用新鲜制备的甲醛还是商业甲醛溶液进行固定，FISH都同样成功。与匀浆器技术相比，手动解剖固定的卵巢管每个成虫的产量要高得多（便于对突变体进行分析），特别是年轻的卵室会更丰富，尽管该过程本身更费力。
