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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Methanol can be used as an auxiliary fixed medium for retinal whole-mount preparations and long-term storage, which is useful for the investigation of retinal ganglion cells.

Abstract

Retinal ganglion cells (RGCs), which are the projection neurons of the retina, propagate external visual information to the brain. Pathological changes in RGCs have a close relationship with numerous retinal degenerative diseases. Whole-mount retinal immunostaining is frequently used in experimental studies on RGCs to evaluate the developmental and pathological conditions of the retina. Under some circumstances, some valuable retina samples, such as those from transgenic mice, may need to be retained for a long period without affecting the morphology or number of RGCs. For credible and reproducible experimental results, using an effective preserving medium is essential. Here, we describe the effect of methanol as an auxiliary fixed medium for retinal whole-mount preparations and long-term storage. In brief, during the isolation process, cold methanol (−20 °C) is pipetted onto the surface of the retina to help fix the tissues and facilitate their permeability, and then the retinas can be stored in cold methanol (−20 °C) before being immunostained. This protocol describes the retina isolation workflow and tissue sample storage protocol, which is useful and practical for the investigation of RGCs.

Introduction

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 t....

Protocol

All steps are performed at room temperature unless otherwise indicated. All C57BL/6J mice used were obtained from the Laboratory Animal Center of Wuhan University, and all the related experiments were approved by the Committee on the Ethics of Animal Experiments of Wuhan University. All efforts were made to minimize the suffering of the mice.

1. Enucleation and fixation of the eyes

  1. Euthanize the mice with carbon dioxide asphyxiation, and enucleate the eyeball gentl.......

Representative Results

After dissection, the retina should look like a flat four-leaf clover. In this study, by using the protocol outlined above, the retina turned white after methanol was added (Figure 1). Meanwhile, the retina changed from soft to pliable and flat. Next, the RGCs were labeled with anti-RBPMS8. Four image fields were taken in the whole-mount retina (n = 3) using a confocal microscope (eyepiece: 10x; objective: 40x). Representative views of visualized RGCs of retinas befor.......

Discussion

Fixation is an essential step to preserve the retina, which can impact any subsequent RGC investigations based on morphology. Successful fixation rapidly captures the structure and state of the retinas at the moment of exposing them to the fixing medium, which is critical for further analysis. Although formaldehyde has been regarded as one of the most common fixing agents for tissue and cell fixation and preservation, formaldehyde alone does not always work well as the optimal chemical fixative for some investigations

Acknowledgements

This work was funded by the Hubei Key Laboratories Opening Project (grant no. 2021KFY055), Natural Science Foundation of Hubei Province (grant no. 2020CFB240), and Fundamental Research Funds for the Central Universities (grant no. 2042020kf0065).

....

Materials

NameCompanyCatalog NumberComments
24-well cell culture clusterCostarEyeball fixation
24-well hemagglutination plateLabedit CompanyIncubation antibody
Adhesion microscope slidesCitotestOr similar
Anti-fluorescent quenching mountantServicebioG1401Slow down fluorescence quenching
BSA (bovine serum albumin)ServicebioGC305010Blocking reagent
Confocal microscopeOLYMPUSApply 40x objective lens
Curved scissorsJiangsu Kanghua Medical Equipment Co., Ltd.Dissecting tools
Dissecting microscopeRWD Life science Co.,LTD 77001SDissecting tools
ForcepsJiangsu Kanghua Medical Equipment Co., Ltd.Dissecting tools
MethanolSinopharm Chemical Reagent Co., Ltd.20210624GC≥99.5%
Nail polishSecheViteSealing agent
Needles Shanghai Kindly Enterprises Development Group Co., Ltd.Accelerate the fixation
Paraformaldehyde solutionServicebioG1101Eyeball fixation
PBS (phosphate buffered saline pH 7.4)ServicebioG0002Rinse the eyeball 
Primary antibody: guinea pig anti-RNA-binding protein with multiple splicing (RBPMS)PhosphoSolutionsCat. #1832-RBPMSFor immunofluorescence. Used at 1:400
Secondary antibody: Cy3 affiniPure donkey anti-guinea pig IgG (H+L)Jackson ImmunoResearch706-165-148For immunofluorescence. Used at 1:400
Straight scissorsJiangsu Kanghua Medical Equipment Co., Ltd.Dissecting tools

References

  1. Sanes, J. R., Masland, R. H. The types of retinal ganglion cells: Current status and implications for neuronal classification. Annual Review of Neuroscience. 38, 221-246 (2015).
  2. Almasieh, M., Wilson, A. M., Morquette, B., Cueva Vargas, J. L., Di Polo, A.

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Retinal Ganglion CellsWhole mount RetinaRetina IsolationMethanol FixationTissue PreservationOptic NeuropathyRetinal Degeneration

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