JoVE Logo
Faculty Resource Center

Sign In





Representative Results






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

Published: April 7th, 2023



1Department of Ophthalmology, Renmin Hospital of Wuhan University

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.

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.

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....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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).


Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
24-well cell culture cluster Costar Eyeball fixation
24-well hemagglutination plate Labedit Company Incubation antibody
Adhesion microscope slides Citotest Or similar
Anti-fluorescent quenching mountant Servicebio G1401 Slow down fluorescence quenching
BSA (bovine serum albumin) Servicebio GC305010 Blocking reagent
Confocal microscope OLYMPUS Apply 40x objective lens
Curved scissors Jiangsu Kanghua Medical Equipment Co., Ltd. Dissecting tools
Dissecting microscope RWD Life science Co.,LTD  77001S Dissecting tools
Forceps Jiangsu Kanghua Medical Equipment Co., Ltd. Dissecting tools
Methanol Sinopharm Chemical Reagent Co., Ltd. 20210624 GC≥99.5%
Nail polish SecheVite Sealing agent
Needles  Shanghai Kindly Enterprises Development Group Co., Ltd. Accelerate the fixation
Paraformaldehyde solution Servicebio G1101 Eyeball fixation
PBS (phosphate buffered saline pH 7.4) Servicebio G0002 Rinse the eyeball 
Primary antibody: guinea pig anti-RNA-binding protein with multiple splicing (RBPMS) PhosphoSolutions Cat. #1832-RBPMS For immunofluorescence. Used at 1:400
Secondary antibody: Cy3 affiniPure donkey anti-guinea pig IgG (H+L) Jackson ImmunoResearch 706-165-148 For immunofluorescence. Used at 1:400
Straight scissors Jiangsu Kanghua Medical Equipment Co., Ltd. Dissecting tools

  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. The molecular basis of retinal ganglion cell death in glaucoma. Progress in Retinal and Eye Research. 31 (2), 152-181 (2012).
  3. Au, N. P. B., Ma, C. H. E. Neuroinflammation, microglia and implications for retinal ganglion cell survival and axon regeneration in traumatic optic neuropathy. Frontiers in Immunology. 13, 860070 (2022).
  4. Pavlidis, M., Stupp, T., Naskar, R., Cengiz, C., Thanos, S. Retinal ganglion cells resistant to advanced glaucoma: A postmortem study of human retinas with the carbocyanine dye DiI. Investigative Ophthalmology & Visual Science. 44 (12), 5196-5205 (2003).
  5. Vidal-Sanz, M., et al. Understanding glaucomatous damage: anatomical and functional data from ocular hypertensive rodent retinas. Progress in Retinal and Eye Research. 31 (1), 1-27 (2012).
  6. Kole, C., et al. Activating transcription factor 3 (ATF3) protects retinal ganglion cells and promotes functional preservation after optic nerve crush. Investigative Ophthalmology & Visual Science. 61 (2), 31 (2020).
  7. Nadal-Nicolás, F. M., et al. Brn3a as a marker of retinal ganglion cells: qualitative and quantitative time course studies in naive and optic nerve-injured retinas. Investigative Ophthalmology & Visual Science. 50 (8), 3860-3868 (2009).
  8. Kwong, J. M., Caprioli, J., Piri, N. RNA binding protein with multiple splicing: A new marker for retinal ganglion cells. Investigative Ophthalmology & Visual Science. 51 (2), 1052-1058 (2010).
  9. Stradleigh, T. W., Ishida, A. T. Fixation strategies for retinal immunohistochemistry. Progress in Retinal and Eye Research. 48, 181-202 (2015).
  10. Stradleigh, T. W., Greenberg, K. P., Partida, G. J., Pham, A., Ishida, A. T. Moniliform deformation of retinal ganglion cells by formaldehyde-based fixatives. Journal of Comparative Neurology. 523 (4), 545-564 (2015).
  11. Bucher, D., Scholz, M., Stetter, M., Obermayer, K., Pflüger, H. J. Correction methods for three-dimensional reconstructions from confocal images: I. Tissue shrinking and axial scaling. Journal of Neuroscience Methods. 100 (1-2), 135-143 (2000).
  12. Chidlow, G., Daymon, M., Wood, J. P., Casson, R. J. Localization of a wide-ranging panel of antigens in the rat retina by immunohistochemistry: Comparison of Davidson's solution and formalin as fixatives. Journal of Histochemistry & Cytochemistry. 59 (10), 884-898 (2011).
  13. Tokuda, K., et al. Optimization of fixative solution for retinal morphology: A comparison with Davidson's fixative and other fixation solutions. Japanese Journal of Ophthalmology. 62 (4), 481-490 (2018).
  14. Miki, M., Ohishi, N., Nakamura, E., Furumi, A., Mizuhashi, F. Improved fixation of the whole bodies of fish by a double-fixation method with formalin solution and Bouin's fluid or Davidson's fluid. Journal of Toxicologic Pathology. 31 (3), 201-206 (2018).
  15. Zanini, C., Gerbaudo, E., Ercole, E., Vendramin, A., Forni, M. Evaluation of two commercial and three home-made fixatives for the substitution of formalin: A formaldehyde-free laboratory is possible. Environmental Health. 11, 59 (2012).
  16. Tang, M., et al. An optimized method to visualize the goblet cell-associated antigen passages and identify goblet cells in the intestine, conjunctiva, and airway. Immunobiology. 227 (6), 152260 (2022).
  17. Brock, R., Hamelers, I. H., Jovin, T. M. Comparison of fixation protocols for adherent cultured cells applied to a GFP fusion protein of the epidermal growth factor receptor. Cytometry. 35 (4), 353-362 (1999).
  18. Baykal, B., Korkmaz, C., Kocabiyik, N., Ceylan, O. M. The influence of post-fixation on visualising vimentin in the retina using immunofluorescence method. Folia Morphologica. 77 (2), 246-252 (2018).
  19. Powner, M. B., et al. Visualization of gene expression in whole mouse retina by in situ hybridization. Nature Protocols. 7 (6), 1086-1096 (2012).
  20. Zhang, N., Cao, W., He, X., Xing, Y., Yang, N. Using methanol to preserve retinas for immunostaining. Clinical and Experimental Ophthalmology. 50 (3), 325-333 (2022).
  21. Kalesnykas, G., et al. Retinal ganglion cell morphology after optic nerve crush and experimental glaucoma. Investigative Ophthalmology & Visual Science. 53 (7), 3847-3857 (2012).
  22. Parrilla-Reverter, G., et al. Time-course of the retinal nerve fibre layer degeneration after complete intra-orbital optic nerve transection or crush: a comparative study. Vision Research. 49 (23), 2808-2825 (2009).

This article has been published

Video Coming Soon

JoVE Logo


Terms of Use





Copyright © 2024 MyJoVE Corporation. All rights reserved