Optimized Protocol for Intestinal Swiss Rolls and Immunofluorescent Staining of Paraffin Embedded Tissue

Sarah A. Dooley; Rachel Stubler; Rachel M. Edens; Piper R. McKee; Jordan N. Rucker; Amy Engevik

doi:10.3791/66977

JoVE Journal > Biology

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Biology

用于肠瑞士卷和石蜡包埋组织的免疫荧光染色的优化方案

Published: July 19, 2024

doi:

10.3791/66977

Sarah A. Dooley*¹, Rachel Stubler*¹, Rachel M. Edens, Piper R. McKee, Jordan N. Rucker, Amy Engevik

¹Department of Regenerative Medicine and Cell Biology,Medical University of South Carolina

Summary

肠道对消化和吸收至关重要。由于独特的细胞结构，每个区域（十二指肠、空肠、回肠、结肠）都发挥着不同的功能。研究肠道生理学需要细致的组织分析。该方案概述了使用 Swiss roll 技术进行组织固定和处理，通过适当的组织保存和定位确保准确的免疫染色。

Abstract

肠道是一个复杂的器官，由小肠和大肠组成。小肠可进一步分为十二指肠、空肠和回肠。肠道的每个解剖区域都有独特的功能，这反映在细胞结构的差异上。研究肠道的变化需要对不同的组织区域和细胞改变进行深入分析。为了研究肠道并观察大块组织，研究人员通常使用一种称为肠道瑞士卷的技术。在这种技术中，肠道被分成每个解剖区域并固定在平坦的方向上。然后，小心地卷起组织并处理石蜡包埋。正确的组织固定和定向是一种经常被忽视的实验室技术，但对于下游分析至关重要。此外，肠道组织不适当的瑞士滚动会损害脆弱的肠上皮，导致免疫染色组织质量差。确保组织固定良好且方向正确、细胞结构完整是确保肠道细胞最佳可视化的关键步骤。我们提出了一种经济高效且简单的方法来制作瑞士卷，将肠道的所有部分包含在一个石蜡包埋块中。我们还描述了肠道组织的优化免疫荧光染色，以研究肠上皮的各个方面。以下方案为研究人员提供了通过肠道组织固定、Swiss-roll 技术和免疫染色获得高质量免疫荧光图像的综合指南。采用这些精细的方法可以保留肠上皮的复杂形态，并促进对肠道生理学和病理生物学的更深入理解。

Introduction

当肠道组织被保存以进行免疫染色时，肠道的细胞结构在维持其结构完整性方面构成了独特的挑战。小肠由细长的指状结构组成，称为绒毛¹。这些绒毛在包埋过程中经常变形。确保研究人员拥有正确嵌入肠道以获得横截面的技术，允许可视化肠道的所有区域以及构成肠道的各层（即固有肌层、粘膜和浆膜），对于稳健的实验分析至关重要²。固定不充分、过度固定和组织处理不当会损害组织完整性，导致对肠上皮的意外损伤 ^3,4。在这些步骤中损伤肠上皮会显着降低后续分析的质量，例如免疫荧光，无论免疫组化方案和所采用的抗体的功效如何。

免疫染色与适当的组织固定一样，是生物医学研究的重要组成部分。如果操作得当，免疫染色可以阐明细胞结构和功能以前未知的方面。由于固定和石蜡包埋过程导致的物理化学修饰，石蜡切片的免疫荧光染色可能具有挑战性⁵。固定和石蜡包埋导致抗原掩蔽，这会干扰目标表位的免疫荧光检测⁶。延迟固定可诱导蛋白水解降解，从而导致关键表位染色减弱或缺失⁷。此外，抗体通常不准确，背景水平高。促进一致和特异性抗体结合以及高信噪比的免疫染色方案可以为研究人员提供有价值的信息。

在这里，我们提供了一个全面的方案，旨在通过肠道组织固定、瑞士卷制备⁸ 和免疫染色获得高质量的免疫荧光图像。该方案强调保护肠道完整性的指南，旨在为研究人员提供一种强大的方法，以提高免疫荧光成像研究的质量和可靠性。我们还寻求使用具有成本效益的资源，包括滤纸和自制抗原修复、封闭溶液和抗体稀释剂，以使可能资金有限的实验室更容易获得该方案。对于所有实验方案，研究人员应根据其实验方法和感兴趣的领域优化当前方案。

Protocol

南卡罗来纳医科大学机构动物护理和使用委员会批准了所有动物护理、维护和治疗。收集成年 C57BL/6J 小鼠（雄性和雌性 3-5 个月大，体重约 30 g）的肠道组织用于本研究。 1. 肠道组织固定小心地从安乐死的小鼠中解剖出整个肠道，并将其放入含有磷酸盐缓冲盐水（PBS）的称重船或培养皿中。从 PBS 中取出肠道，用剪刀轻轻去除多余的脂肪和结缔…

Representative Results

如前所述，进行苏木精和伊红（H&E）染色12。使用优化的方法，瑞士肠卷在单个载玻片上包括小肠和大肠的所有三个段。将整个肠道放在载玻片上可以让研究人员分析肠道所有部分的变化，并节省切片和染色试剂的成本（图 1）。此外，在免疫染色时将所有肠段同时暴露于相同的溶液中有助于确保准确的结果。H&E 显微照片显示小肠和大肠所有部分的肠道结…

Discussion

在这里，我们提出了一种使用 Swiss roll 技术进行组织固定的优化方法，以保留肠道结构并促进准确的免疫染色。一旦掌握，这项技术可用于研究涉及肠道生理学和细胞生物学的各种研究问题¹⁹。几种优化的瑞士轧制方法已经发布，非常有用^20,21。这种技术的优点是易于在滤纸上准确打开肠道。这使得组织可以固定平整，防止组织在滚动?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项研究得到了美国国立卫生研究院（NIH）对ACE的K01 DK121869资助的支持，而这个出版物部分得到了T32 GM132055，F31 DK139736，T32 DK124191，TL1 TR001451，UL1 TR001450，以及HCS对SAD和RS的基石资助的支持。这项工作得到了南卡罗来纳医科大学（MUSC）到 ACE 的启动资金的支持，并得到了 MUSC 消化疾病研究核心中心（P30 DK123704）和消化和肝病 COBRE （P20 GM120475）的支持。使用 MUSC 的细胞和分子成像核心进行成像。

Materials

β-CATENIN	GeneTex	GTX101435
Cellulose filter paper	Cytiva	10427804	Thick Whatman paper
Charged glass slides	Thermo Fisher Scientific	23888114
Coverslip	Epredia	152440
Dissecting pins size 00	Phusis	B082DH4TZF
E-CADHERIN	R&D Systems	AF748
Freezer gloves	Tempshield	UX-09113-02
Heating block	Premiere	XH-2001	Slide Warmer
Histo-Clear II	Electron Microscopy Sciences	64111-04	Clearing reagent
Hoescht	Thermo Fisher Scientific	62249
Hydrochloric Acid	Sigma Aldrich	320331
Hydrophobic pen	Millipore	402176
LAMININ	GeneTex	GTX27463
LAMP1	Santa Cruz	SC-19992
Large cassettes	Tissue-Tek	4173
Minutien pins	Fine Science Tools	NC9679721
Mouse-on-mouse blocking reagent	Vector Laboratories	MKB-2213	Mouse-on-mouse block
MUC2	GeneTex	GTX100664
PCNA	Cell Signaling Technology	2586S
Pressure Cooker	Cuisinart	B000MPA044
ProLong gold antifade	Thermo Fisher Scientific	P36934	Mounting medium
Reverse action forceps	Dumont	5748
Slide Rack	Tissue-Tek	62543-06
Slide Staining Set	Tissue-Tek	62540-01	Solvent Resistant Dishes and Metal Frame
Small cassettes	Fisherbrand	15-200-403B
Sodium citrate dihydrate	Fisher Bioreagents	BP327-1
Teleostein Gelatin	Sigma	G7765	Blocking buffer
Triton X-100	Thermo Fisher Scientific	A16046
Tween 20	Thermo Fisher Scientific	J20605-AP
Wipes	KimTech	34155
Xylenes	Fisher Chemical	1330-20-7
γ-ACTIN	Santa Cruz	SC-65638

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Dooley, S. A., Stubler, R., Edens, R. M., McKee, P. R., Rucker, J. N., Engevik, A. Optimized Protocol for Intestinal Swiss Rolls and Immunofluorescent Staining of Paraffin Embedded Tissue. J. Vis. Exp. (209), e66977, doi:10.3791/66977 (2024).