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

Выделение сетчатки мыши и лечение метанолом для исследования ганглиозных клеток сетчатки

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.

В центре внимания автора: Цельный препарат на основе метанола для исследования ганглиозных клеток сетчатки  

Цельный препарат на основе метанола для исследования ганглиозных клеток сетчатки

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  

Изоляция и уплощение сетчатки мыши

После фиксации энуклеированного глазного яблока усыпленной мыши перенесите его на предметное стекло и поместите под препарирующий микроскоп. С помощью щипцов зажмите край роговицы, чтобы предотвратить движение глазного яблока. С помощью рассекающих ножниц разрезают вокруг роговицы на глубине примерно один миллиметр через роговицу склерального лимба, а затем удаляют хрусталик и стекловидное тело.Держа диск зрительного нерва в центре, разрезайте сетчатку радиально вдоль четырех квадрантов, достигая примерно 2/3 радиуса сетчатки. Зажмите один из краев склеры с помощью зубчатых щипцов. Удерживайте край склеры незубчатыми щипцами и двигайтесь от зубчатых щипцов к основанию склеры.Аккуратно очистите сетчатку. Смочите сетчатку 200 микролитрами PBS с помощью пипетки. Удалите излишки PBS с помощью впитывающей бумаги и при необходимости разгладьте сетчатку с помощью небольшой щетинной щетки.Медленно добавляйте холодный метанол по каплям на внутреннюю поверхность сетчатки, пока она полностью не покроется. Как только сетчатка побелеет и разовьет повышенную прочность, аккуратно сплющите сетчатку и удалите загрязнения, такие как остаточные пигментные мембраны и стекловидное тело, с помощью небольшой щетинистой щетки. После инвертирования сетчатки обработайте ее метанолом, как было продемонстрировано ранее, и перенесите ее в лунку с 24-луночной пластиной, замочите ее в метаноле и храните при температуре 20 градусов Цельсия в течение одного-двух часов для немедленного иммуноокрашивания.Затем промойте сетчатку PBS после удаления метанола из пластины. Ретиноловые ганглиозные клетки сетчатки до и после лечения метанолом визуализировали с помощью конфокальной микроскопии, которая показала, что 12 месяцев консервации метанола не повлияли на иммуноокрашивание RGC.

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 ...