后期阶段的胚胎和幼虫的超声，促进免疫荧光染色<em>果蝇</em>组织<em>原位</em

Summary

Immunostaining is an effective technique for visualizing specific cell types and proteins within tissues. By utilizing sonication, the protocol described here alleviates the need to dissect Drosophila melanogaster tissues from late-stage embryos and larvae before immunostaining. We provide an efficient methodology for the immunostaining of formaldehyde-fixed whole mount larvae.

Abstract

在果蝇进行研究果蝇胚胎和幼虫提供重要的洞察，如细 ​​胞命运规范和器官发育过程。免疫允许开发组织和器官的可视化。然而，保护角质层形成于胚胎发育的最后阻止抗体渗透到后期的胚胎和幼虫。虽然之前的免疫清扫经常用来分析果蝇幼虫组织，它证明效率低下的一些分析，因为小组织可能难以定位和分离。超声提供了果蝇幼虫的免疫方案替代清扫。它允许大量的后期胚胎和幼虫的快捷，同时处理和维护现场形态。固定在甲醛后，将样品进行超声处理。样品然后进行免疫染色与抗原特异性初级蚂蚁ibodies和荧光标记的第二抗体，通过荧光显微镜可视化靶细胞类型和特定的蛋白质。在超声处理的过程中，超声处理探针的样品上面，以及超声处理的持续时间和强度，正确放置是关键的。可能需要高品质的污渍产生额外稍做修改标准的免疫染色的协议。对于抗体具有低的信噪比，更长的孵育时间通常是必要的。至于概念，这种超声促进的协议的证明，我们将展示三种组织类型（睾丸，卵巢，和神经组织）免疫组化染色，在一系列的发展阶段。

Introduction

果蝇胚胎和幼虫为研究发育过程中的许多器官和组织一个很好的典范。各个单元的成像是在这些研究中常常需要以确定在哪些细胞发育的复杂的环境。细胞在组织中的可视化可以通过免疫染色来完成。以及描述的免疫染色协议果蝇胚胎组织<后17小时产蛋^（AEL）1-3存在。然而，保护表皮的形式朝向胚的末端，以防止有效抗体渗透。因此，这些免疫染色方案是低效的中后期胚胎组织的分析和在幼虫发育（ ^第一龄（L1）， ^第二龄（L2）和^第三龄（L3））的后续阶段。这种低效率施加了障碍，我们在这延长的发展阶段⁴所发生的动态过程的理解。 TISSUE夹层是一种广泛使用的技术来绕过这一障碍^5-7。然而，夹层可以证明效率低下。提取可以通过难以定位或分离胚胎和幼虫组织缠身。此外，物理去除目标组织可以通过破裂它们或通过不提取它们以其整体造成损坏。

超声是采用声波干扰分子间相互作用的方法。它已被用于以免疫染色显影神经细胞类型⁶打乱果蝇幼虫角质层的完整性。这个协议已经适应免疫染色后期胚胎和幼虫性腺，它可以小到50微米的直径^8-10。通过这样的研究中，雄性生殖系干细胞（GSC）利基形成过程的特点在后期果蝇胚胎^8-10和机制调节干细胞的发育和DIFferentiation晚期胚胎性腺和幼虫已经阐明^9-12。因此，超声处理提供了一种有效的替代组织剥离，可能是由于组织大小困难。此外，它使果蝇组织中的原位免疫染色，而使整个有机体的范围内的细胞和保持在原位的形态。在这里，我们描述了通过早期/中期三级组织原位一步一步的协议中晚期胚胎荧光免疫染色。 果蝇性腺和神经组织的分析显示，在代表性的成果来证明这个协议的效力。此外，这种免疫方案可适于分析其他果蝇组织以及组织中其他生物具有外表皮。

Protocol

1，准备集合笼麻醉年轻，肥沃的苍蝇与CO 2。转让麻醉苍蝇笼子里。为了获得最佳的收益率，用100-120成蝇从2-7日龄以4:1的比例女性为男性。让苍蝇适当的适应期，〜24小时之前获得的样品进行固定。如果笼成立了处女的女性交配雄性，一个用36 – 48小时的适应期。 在笼的开放端，将事先准备好的苹果汁琼脂平板上用酵母膏中的中心在保持器的开口部的硬币大小的滴。然后，…

Representative Results

为了证明在晚期胚胎和原位幼虫组织分析超声系免疫的功效，野生型胚胎和幼虫处理用于免疫睾丸，卵巢，和神经组织。样品通过共聚焦显微镜成像和代表性结果示（ 图1和图2）。结果表明，所描述的协议是有效的用于可视化的形态特征以及各个细胞在原位时果蝇发育的晚期胚胎通过早期/中期L3阶段。 结果从睾丸免疫组织?…

Discussion

这个协议提供了一种方法来成功地免疫组织化学染色的目标果蝇胚胎和幼虫组织原位 ，从而省去了清扫。按照现有协议进行染色早期胚胎^1,2,3，绒毛膜膜是用50％的漂白剂（次氯酸钠）除去。样品被固定在甲醛和甲醇。因为幼虫角质层导致老年人样品浮起，将整个样品然后通过细胞过滤器传递到保证幼虫保留。样本被存储，如果需要的话，在化学级甲醇。补液后，正确实施超…

Materials

			Table 1: Reagents and Buffers
Phosphate Buffer Triton X-100 (PBTx)	For 5 L: 500 mL PBS 10X 4.45 L ddH2O 50 mL Triton 10%	Store at room temperature.
Phosphate Buffer Saline 10x (PBS 10X)	For 1 L in dH2O: 80 g NaCl 2 g KCl 14.4g Na2HPO4 2.4 g KH2PO4	Add components and fill to appropriate volume. Store at 4 degrees C.
Triton 10%	For 50 mL: 5 mL of Triton 5 mL of PBS 10X 45 mL of ddH2O	Rock to mix. Store at room temperature.
PEMS	0.1 M Pipes (pH 6.9) 2.0 mM MgSO4 1.0 mM EGTA	Store at room temperature.
Pipes	For a 400ml of a 0.25 M solution (pH 6.9): 30.24 g Pipes dH20 NaOH	Dissolve Pipes in 300 mL dH2Oand then adjust to pH 6.9 with NaOH. Bring the total volume to 400 mL with dH2O and autoclave. Store at room temperature.
Formaldehyde	37% formaldehyde by weight in methanol	Store at room temperature. Store formaldehyde, heptane, and methanol waste mixture in a tightly sealed container in fume hood before disposal as per institutional guidelines.
Heptane	n-Heptane	CAS 142-82-5. Store at room temperature. Store formaldehyde, heptane, and methanol waste mixture in a tightly sealed container in fume hood before disposal as per institutional guidelines.
Methanol	Methanol	CAS 67-56-1. Store at room temperature. Store formaldehyde, heptane, and methanol waste mixture in a tightly sealed container in fume hood before disposal as per institutional guidelines.
Phosphate Buffer Tween (PBTw)	To make 1 L: 100 mL PBS 10X 890 mL ddH2O 10 mL Tween 10%	Filter sterilize after adding all components. Store at 4 degrees C.
Tween 10%	For 50 mL: 5 mL Tween 5 mL of PBS 10X 40 mL of ddH2O	Rock to mix. Store at room temperature.
Bovine Serum Albumin/Phosphate Buffer Tween (BBTw)	To make 1 L: 100 mL PBS 10X 890 mL ddH2O 10 mL Tween 10% 1 g Bovine Serum Albumin (BSA)	Add BSA then sterilize using a 0.2 micrometer vacuum filter unit. Store at 4 degrees C.
Normal Goat Serum (NGS)	To make 10 mL: Normal Goat Serum (Jackson ImmunoResearch Laboratories Code: 005-000-121) 10 mL ddH2O	Add ddH2O to vial of NGS and sterilize using a 0.2 micrometer syrninge filter. Store aliquots at -20 degrees C.
1,4-diazabicyclo[2.2.2]octane (DABCO)	To make 100 mL: 25 mL ddH2O 1 mL Tris HCl (1M, pH 7.5) 2.5 g of DABCO solid (CAS: 281-57-9) 3.5 mL 6N HCl 250 uL 10N NaOH 70 mL glycerol	In 250 mL beaker with stir bar, add ddH2O, Tris HCl and DABCO. Stir and then add 6N HCl, 10 N NaOH, and glycerol. Then add 10NaOH dropwise until solution reaches pH 7.5. Aliquot. Store aliquots at -20 degrees C.
DABCO + p-phenylenediamine (PPD) Solution	1.765 mL NaHCO3 0.353 Na2CO3 0.02 g PPD (CAS: 106-50-3)	Dissolve PPD in NaHCO3 and NaCO3 solution. Add 60 uL of PPD solution to 500 uL of DABCO. Store aliquots at -20 degrees C.
Apple juice plates	To make ~200 plates: 45 g agar (CAS#9002-18-0) 45 g granulated sugar (store bought) 500 mL Apple juice (store bought) 15 mL Tegosept 10% 1.5 mL ddH2O	Add agar to ddH2O in 4L flask then autoclave for 30 minuntes. Mix apple juice and sugar on heated stir plate. Gradually add apple juice mixture to autoclaved agar. Mix on heated stir plate then aliquot 10mL volumes into 35 mm petri dishes and let stand at room temperature to solidfy. Store at 4 degrees C.
Tegosept 10%	To make 100mL: 10g Tegosept 100 mL Ethanol	Store aliquots at -20 degrees C
Yeast paste	~50 g Dry Active Yeast	Gradually add ddH2O to beaker containing yeast while stirring until paste-like consistency reached. Store at 4 degrees C.
			Table 2: Staining Materials
DAPI	1:1000	Invitrogen	D3571 //// Stock at 5mg/mL
rabbit anti-Vasa	1:250		A gift from Ruth Lehmann
mouse anti-Fasciclin III	1:10	Developmental Studies Hybridoma Bank (DSHB)	7G10
mouse anti-1B1	1:4	Developmental Studies Hybridoma Bank (DSHB)	1B1
guniea pig anti-Traffic Jam	1:2500		A gift from Dorthea Godt (Li et al, 2003)
mouse anti-Prospero	1:10	Developmental Studies Hybridoma Bank (DSHB)	Prospero MR1A
rat anti-Elav	1:30	Developmental Studies Hybridoma Bank (DSHB)	Rat-elav 7EBA10 anti-elav
mouse anti-Repo	1:10	Developmental Studies Hybridoma Bank (DSHB)	8D12 anti-Repo
goat anti-rabbit Alexa546	1:500	Invitrogen	A11010
goat anti-mouse Alexa488	1:500	Invitrogen	A11029
goat anti-guniea pig Alexa633	1:500	Invitrogen	A21105
goat anti-rat Alexa488	1:500	Invitrogen	A11006
			Table 3: Materials and Equipment
Fly Cages	Hand-made; Genesee Scientific Corporation	Not applicable; Bottles: 32-130; Pre-made cage: 59-101	Made by cutting clear cast acrylic tubing (1 3/4 inch in diameter) into 4 inch tall segments with a compound miter saw at 400 rpm. Ultrafine stainless steel screening (was attached to one end of the tub with acrylic compund glue. An alternate method using an empty fly food bottle can be found in Drosophila Protocols ISBN 0-87969-584-4. Cages may also be purchased from the Genesee Scientific Corporation.
Sonicator: Branson 250 Digital Sonifier	Branson	Model: Branson Digital Sonifier 250
Sonicator Probe	Branson	Model #: 102C (CE)	EDP: 101-135-066; S/N: OBU06064658
Syringe filter	Nalgene	190-25-20	0.2 micrometer cellulose, acetate membrane filter
Imaging system: Spinning disc confocal microscope with multichromatic light source, digital CCD camera, and imaging software	Microscope: Olympus Light source: Lumen Dynamics Camera: Q-Imaging Imaging Software: Intelligent Imaging Inc.	Microscope: BX51 equipped with DSU spinning disc Light source: X-Cite 120Q Camera: RETIGA-SRV Imaging Software: Slidebook 5.0
Vacuum filter unit	Nalgene	450-0020	0.2 micrometer cellulose nitrate membrane filter