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

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 RGCs^2,3. Analyzing the morphological and quantitative changes of RGCs is a crucial step in determining how neurodegenerative diseases develop and advance^4,5.

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 on^7,8. 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 tissue^9,10.

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 cells^9,10. Different retinal fixation treatments may affect the retinal structure, protein immunogenicity, fluorescence excitation , and attenuation quenching cycle differently^12,13. 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.

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 gently using toothless tweezers.
2. Rinse the eyeball once in phosphate-buffered saline (PBS).
3. Puncture the eyeball with a syringe needle at the cornea under a microscope (eyepiece WF: 10x/22; continuous magnification objective: 0.67x-4.5x) to accelerate the fixation into the eyeball and improve the fixing efficiency.
4. Transfer to a 24-well plate containing 4% paraformaldehyde (PFA).
5. Fix the eyeball in 4% PFA for 45 min, and then transfer it to 1x PBS to remove excess PFA (wash three times, 5 min/time) immediately.

2. Retina isolation and flattening

1. Transfer the eyeball to a glass slide, and place it under a dissecting microscope (eyepiece WF: 10x/22; continuous magnification objective: 0.67x-4.5x).
2. Hold the forceps in one hand, and use it to clamp the margin of the cornea to prevent eyeball movement. Use the other hand to hold the dissecting scissors and cut around the cornea at approximately 1 mm of depth through the corneoscleral limbus. Remove the lens and vitreous body.
3. Hold the optic disc at the center, and using dissecting scissors, cut the retina radially along the four quadrants, reaching approximately 2/3 of the radius of the retina.
4. Clamp one of the scleral edges using toothed forceps. With the other hand, hold the edge of the sclera with untoothed forceps, and move from where the sclera is fixed by the toothed forceps toward the base of the sclera. Carefully peel the retina.
5. Draw off PBS using a pipette (200 µL) to flush and wet the retina, and then remove any excess PBS with a small piece of absorbent paper. If needed, gently flatten the retina with a small bristle brush.
6. Slowly pipette cold methanol (precooled in a −20°C freezer) drop by drop onto the inner surface of the retina (the volume of methanol is not fixed, as long as the retina can be completely coveredby the methanol). The retina turns white and develops increased tenacity.
7. Gently flatten the retina, and remove impurities such as residual pigment membranes and the vitreous body with small bristle brushes. Invert the retina using small bristle brushes, and repeat the above-mentioned process.
8. Transfer the methanol-treated retina into a well of a 24-well plate, and keep it soaked in methanol.
9. Store it at −20°C for 1-2 h for immediate immunostaining, or leave the retina in methanol, and store it at −20°C for long-term storage.
10. Remove the methanol from the 24-well plate, and rinse the retina with 1x PBS.

3. Immunofluorescent staining of the RGCs

1. Carefully transfer the retina to a 24-well hemagglutination plate with a plastic Pasteur pipette, and block in 5% PBS-TX-BSA (5 g of bovine serum albumin powder dissolved in 100 mL of 1x PBST solution; the PBST is formed by adding 10 mL of Triton X-100 to 2,000 mL of 1 x PBS) at room temperature for 4 h or overnight at 4 °C. Keep the ganglion side up.
2. Remove the PBS-TX-BSA, and incubate the retina at 4 °C for 48-96 h, adding 100 µL of the selected primary antibody (guinea pig anti-RBPMS antibody) using a 1:400 dilution.
3. Remove the primary antibody, and rinse the retina three times (20 min each) with 1x PBS, shaking at room temperature.
4. Incubate the retina with gentle shaking overnight at 4 °C with 100 µL of the corresponding secondary antibody (see Table of Materials), cyanine Cy3 affiniPure donkey anti-guinea pig IgG (H+L), using a 1:400 dilution.
5. Rinse the retina with 1x PBS three times (20 min each).
6. Lay the retina flat on the slide with the ganglion cell layer facing upward, drop an appropriate amount of anti-fluorescence quenching agent around the retina with a pipette, and cover the slide with a coverslip (be careful not to have bubbles).
   NOTE: To determine the appropriate amount of anti fluorescence quencher, ensure that it covers the entire retina. After that, cover the slide without allowing the retina to float and move easily.
7. Seal the slide all around with a sealing agent to prevent it from moving. Store the samples at −20°C, and protect them from light.
8. Image the retina using a fluorescence/confocal microscope.

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

Materials

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