Isolation of Intact Eyeball to Obtain Integral Ocular Surface Tissue for Histological Examination and Immunohistochemistry

Dianlei Guo; Chunqiao Liu

doi:10.3791/60086

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Biology

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JoVE Journal Biology

Isolation of Intact Eyeball to Obtain Integral Ocular Surface Tissue for Histological Examination and Immunohistochemistry

分离完整眼珠以获得整体眼表面组织，用于组织学检查和免疫组织化学

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05:15 min

October 20, 2019

DOI: 10.3791/60086-v

Dianlei Guo¹, Chunqiao Liu¹

¹State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center,Sun Yat-sen University

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Overview

This protocol focuses on a method for isolating the mouse eyeball with its eyelid and ocular surface intact, preserving its anatomy for anatomical studies. The technique is particularly beneficial for ophthalmologists interested in examining ocular surfaces without damaging delicate tissues.

Key Study Components

Research Area

Ocular Development
Ocular Diseases
Histology and Immunostaining

Background

Importance of preserving ocular surface integrity
Challenges in dissecting mouse eyeballs without damage
Applications for ophthalmological education

Methods Used

Sequential removal of skull and orbital bones
Mouse model
Use of paraformaldehyde for tissue fixation

Main Results

The protocol preserves the ocular surface epithelial sheet for further examination
Enables histological or immunostaining analysis
Demonstrates effective techniques for ocular dissection

Conclusions

The study showcases a reliable method for isolating the mouse eyeball with attached structures
Significant for studies related to ocular health and diseases

Frequently Asked Questions

What is the primary goal of this protocol?

The primary goal is to isolate the mouse eyeball and its surrounding tissues in an intact state for further biological examination.

Who can benefit from this protocol?

Ophthalmologists and researchers interested in ocular surface studies can benefit the most from this protocol.

What preservation method is used in this protocol?

The tissues are prefixed using 4% paraformaldehyde to preserve their structure before further analysis.

How does the method avoid tissue damage?

Careful dissection and removal of facial bones minimize damage to the ocular tissues.

What are the final applications of the dissected eyeball?

The dissected eyeballs can be used for histological analysis and immunostaining studies.

Is prior experience necessary to perform this protocol?

Some laboratory experience with dissection techniques may be beneficial but is not strictly necessary.

Are there specific tools recommended for this dissection?

Sharp scissors and forceps are recommended to facilitate the dissection process and maintain tissue integrity.

该协议描述了一种在相对完整位置分离眼睑、眼表面、前段和后段的小鼠眼球的方法。

我们的实验室专注于眼部发育和疾病。该协议描述了一种从解剖的小鼠头部中按顺序取出头骨和轨道骨骼的方法。将眼睑、眼表面、透镜和视网膜放在一起。该协议将有利于对眼表面教学感兴趣的眼科医生。我是郭迪安，在解剖面部骨骼时，在试图避免眼球的损害时，告诉技术可能挣扎。这种解剖物质的主要优点是，眼表面上皮板的完整性保存完好，眼表面的不同部分可以通过组织学或免疫覆盖在一个部分检查。一定要切断软组织的所有小骨头和碎屑。原因，它会导致疤痕标记组织部分，否则。对鼠标进行安乐死后，用一把直剪刀切断头部，然后沿着下垂中线一样，从腹腔骨到鼻腔。将头的两半放入干净的培养皿中，用锋利的剪刀切断下颚和舌头。释放视神经，在视光基亚姆位置之前切断它的大脑。并删除所有脑组织，包括嗅觉球，这是连接到头骨壁。现在，所有剩余的组织应有与眼球相关的所有头骨和轨道骨骼。用 PBS 清洗其余部分，以清除任何血液和头发碎屑。将组织放在含有4%甲醛的50毫升管中，并在室温下旋转，以预先固定。前缀完成后，快速洗涤PBS中的解剖头三次，以进一步消除破碎的头发和固定。将组织放入10厘米的培养皿中，沿着文本协议图之一所示平面将头骨切成三个部分。眼球和插座隐藏在头骨的中间部分，在头骨下，正面和骨骼。使用两对四号直钳剥离 ethmoid 骨骼，并完全露出正面和最大骨骼。然后，在地理上将头骨表面划分为四个方面。将弯曲钳尖水平插入每个区域，以按顺序去除最大骨骼和正面骨骼。在此之后，底层的拉克里姆和巨骨将暴露。去除眼球周围的两块骨头和相关皮下肌肉和脂肪。将眼睑和皮肤附着的眼球修剪成一个方形的小方块，以减少组织体积，并在嵌入时方便组织定向。将眼球和相关组织放回4%的甲醛中，并在4点继续通宵修复

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