Name: Mouse Retina Isolation and Flattening
Uploaded: 2023-04-07T12:20:00
Description: Watch this Scientific Journal Video about Mouse Retina Isolation and Flattening at JoVE.com

mouse retina isolation and flattening

망막 신경절 세포 연구를 위한 마우스 망막 분리 및 메탄올 치료

Retinal ganglion cells (RGCs) are the only projection neurons in the retina, and they integrate and transmit outside visual information to the brain1. Many neurodegenerative diseases such as glaucoma and traumatic optic neuropathy are characterized by irreversible damage and loss of RGCs2,3. Analyzing the morphological and quantitative changes of RGCs is a crucial step in determining how neurodegenerative diseases develop and advance4,5.
Indirect immunofluorescence assay is a widely accepted method to monitor the distribution of proteins and cell counting. In the laboratory, whole-mount retinal immunostaining is commonly used in experimental studies on RGCs to evaluate the physiological and pathological conditions of the retina6. The most common markers used for RGC quantification in the whole retina include Brn3a, RNA binding protein with multiple splicing (RBPMS), and so on7,8. Characterizing the amount and distribution of RGCs requires high-quality whole-mount retinal immunostaining. Generally, in immunostaining protocols, the retina is immersed in chemical fixatives before being incubated in antibodies. Ideal fixatives should not change the shape of cells, the accessibility or affinity of the epitopes for antibodies, or the linear dimensions of the tissue9,10. 
Due to the complex structure of the retina, problems such as retinal fragility and folding, as well as some common difficulties including cell shrinkage and unclear nuclei, are prone to occur when making a whole-mount retinal patch, which has a negative impact on experimental research. In addition, not all retinas are immunostained immediately, especially when it comes to the retinas of transgenic mice with expensive prices that are of precious origin, necessitating the preservation of the extra retinal samples for further use.
The appropriate fixative solution can fix the tissue quickly, avoid tissue autolysis, preserve the normal morphology and structure of the tissue cells, and keep the antigenicity of proteins and other substances10. At present, formaldehyde-based fixation has been widely used in various tissues, including separated retinas, hemisected eyecups, and whole eyeballs10. Tissue shrinkage and the morphological alteration of cells are the two critical challenges encountered following immersion in formaldehyde11. In addition, modified fixation formulations are increasingly emerging to maximize the retention of the original properties of the retina and the target cells9,10. Different retinal fixation treatments may affect the retinal structure, protein immunogenicity, fluorescence excitation , and attenuation quenching cycle differently12,13. Retinas fixed with Davidson's solution are more morphologically intact compared to those fixed with formalin, but Davidson's solution is less compatible with some antibodies, such as microglial marker-ionized calcium binding adaptor molecule 112. Considering the fragile nature of retinas, researchers would naturally wonder whether the retinal integrity, as well as the properties and morphology of the target cells, will change after long-term storage. However, the possible effects of fixation solution on retinal and RGC cell morphology after storage for several months have rarely been reported. The optimization of retinal fixation is critical for the evaluation and preservation of RGCs. 
We provide a detailed description of a reliable and technically straightforward method that we use for whole-mount murine retinal staining. Our method emphasizes the proper preparation and storage of retinas for RGC investigation, taking into account the need for the long-term storage of retinal tissue as well as specific aspects of fluorophore formation or degradation.

저자 스포트라이트: 망막 신경절 세포 연구를 위한 메탄올 기반 전체 장착 준비  

망막 신경절 세포 조사를 위한 메탄올 기반 전체 마운트 준비

The research aims to provide a modified retina isolation workflow and tissue sample storage protocol, which is useful and practical for investigating RGCs. Retinas are fragile and folding, which limits experimental research when making a whole-mount retinal patch. In addition, all retinas do not require immediate immunostain.Sometimes, extra retina samples need preservation for further use. We provide a detailed description of a reliable and technically straightforward method of whole-mount retina stain that emphasizes the proper preparation and the need for the long-term storage of retinas for RGC investigation. We will continue to work on the basic research of degenerative optic neuropathies.Further, the molecular mechanism of optic nerve injury and accident regeneration and potential clinical targets for diagnosing and treating neurodegenerative disease will be explored.

Watch this Scientific Journal Video about Mouse Retina Isolation and Flattening at JoVE.com

Mouse Retina Isolation and Flattening  

마우스 망막 분리 및 평탄화

안락사된 쥐의 적출된 안구를 고정한 후 유리 슬라이드에 옮겨 해부현미경으로 관찰한다. 집게를 사용하여 안구 움직임을 방지하기 위해 각막의 가장자리를 조입니다. 해부 가위로 각막 공막 윤부를 통해 약 1mm 깊이로 각막 주위를 자른 다음 수정체와 유리체를 제거합니다.시신경을 중앙에 두고 4개의 사분면을 따라 방사상으로 망막을 절단하여 망막 반경의 약 2/3에 도달합니다. 톱니 집게를 사용하여 공막 가장자리 중 하나를 클램프하십시오. 톱니가 없는 집게로 공막 가장자리를 잡고 톱니가 있는 집게에서 공막 바닥으로 이동합니다.망막을 조심스럽게 껍질을 벗기십시오. 피펫을 사용하여 200 마이크로리터의 PBS로 망막을 적십니다. 흡수성 종이를 사용하여 과도한 PBS를 제거하고 필요한 경우 작은 강모 브러시로 망막을 평평하게 합니다.차가운 메탄올이 완전히 덮일 때까지 망막의 내부 표면에 천천히 한 방울씩 추가합니다. 망막이 하얗게 변하고 끈기가 증가하면 망막을 부드럽게 평평하게 만들고 작은 강모 브러시로 잔류 색소 막 및 유리체와 같은 불순물을 제거합니다. 망막을 뒤집은 후, 이전에 입증 된대로 메탄올로 처리하고, 24 웰 플레이트의 웰로 옮기고, 메탄올에 담그고 즉각적인 면역 염색을 위해 섭씨 20도에서 1-2 시간 동안 보관한다.그런 다음 플레이트에서 메탄올을 제거한 후 PBS로 망막을 헹굽니다. 메탄올 처리 전후의 망막의 레티놀 신경절 세포를 공초점 현미경을 사용하여 시각화하였으며, 이는 12개월의 메탄올 보존이 RGC 면역염색에 영향을 미치지 않음을 나타냅니다.

Research

JoVE Journal

Neuroscience

Mouse Retina Isolation and Flattening

Methanol-Based Whole-Mount Preparation for the Investigation of Retinal Ganglion Cells

Retinal ganglion cells (RGCs), which are the projection neurons of the retina, propagate external visual information to the brain. Pathological changes in ...