Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Summary

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

Abstract

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.

Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

Introduction

Performing immunohistochemistry in delicate intact tissues runs the risk of damage during handling and transfer. This can occur either in brain slices or other thin tissue, such as degenerated retina. Additionally, there are certain methods of tissue fixation that can be advantageous for immunostaining of neuronal structures, but result in compromised structural stability, precluding their use. A particular example of this is carbodiimide-based fixation, which is superior for staining receptors and hormones^1-7 but is commonly avoided due to the instability of the fixed tissue.

Here, we describe a procedure which utilizes a hydrophilized PTFE membrane to structurally support delicate tissue, either fixed or unfixed, for a variety of staining techniques. The supporting membrane allows for the manipulation of delicate tissue before and after fixation, allowing for several steps of processing while minimizing the risk of tissue damage. Overall, this simple method to preserve tissue integrity provides the opportunity to use techniques that might otherwise be avoided. As such, this approach could also be successfully used for preparation of wide variety of tissues such as brain slices that become fragile following slicing procedures.

Protocol

In all experimental procedures, animals were treated according to the regulations in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and in compliance with protocols approved by Weill Cornell Medical College. Animals were euthanized by carbon dioxide and subsequent cervical dislocation.

1. Dissection of Wholemount Retina

1. After the mouse is euthanized, enucleate its eyes and place them into oxygenated HEPES-buffered Ringer's solution (Table 1).
2. Remove the cornea by cutting in a circular path along the ora serrata with small scissors, holding the eye at the limbus with forceps. Pull out the lens with forceps.
3. Remove as much vitreous as possible, using blunted-edge forceps. This step is important for bolus injections and electrophysiological recordings; it is not necessary for when performing immunohistochemistry alone.
4. Detach the retina from the eyecup by positioning forceps between the retina and the eyecup, moving the forceps slowly around the circumference. Stabilize the eye cup by holding the sclera firmly with a second set of forceps. Cut the optic nerve at the location between the retina and the eyecup to ease the separation of the retina from the eyecup.
5. The retina can be dissected into pieces (e.g. quarters) or used as a whole. To cut into pieces, use scissors to make incisions from the retinal periphery all the way down to the optic nerve. Alternatively, to use the entire retina, make incisions from the periphery halfway to the optic nerve. The incisions will help to make a flat preparation.

2. Mounting onto Hydrophilized PTFE Membrane Insert

1. Prepare the membrane as described below. When physiological recordings or dye injection experiments are performed, shorten the holder by using either a lathe or a handsaw so that the remaining ring has a height of 1 mm.
   Note: The culture plate inserts may be either unmodified or modified (Figure 1A). The choice of the configuration is dictated by the purpose of the experiment. For immunostaining, an unmodified insert is suitable.
2. Polish down rough surfaces with fine (300 grit or higher) sandpaper.
3. (Optional) Remove the feet underneath the ring with scissors.
4. To remove any debris, rinse or sonicate the membrane in distilled water for 2-3 min.
5. Mount the tissue onto the membrane (Figure 1A). Place the tissue with some solution on top of the membrane with a plastic transfer pipette. Cut off the narrow tip of the pipette if necessary, to avoid having the tissue sample adhere to the pipette during transfer.
6. Attach the retina with ganglion cells (GCs) up: on the membrane, gradually remove solution while spreading the retina and unfolding the folds with forceps.
7. On the opposite side of the membrane, position an empty syringe (1 ml) directly underneath the tissue. Apply suction by pulling back on the plunger to a volume of 0.2-0.3 ml. Increase the surface area of suction by cutting the holding tip of the syringe prior to use. The tissue does not require further treatment for adhesion, as it is physically enmeshed within the membrane.

3. Bolus Injection of Isolectin Alexa 488

1. Prepare patch pipettes and staining solution. Pull patch pipettes from borosilicate capillary glass with a Flaming/Brown puller, with a resistance of ~1 MΩ. Gently break the tip of the pipette.
2. Make staining solution by adding 0.3 μl of isolectin Alexa 488 stock solution to 10 μl of the HEPES-buffered Ringer's solution described in step 1.2.
3. Transfer sample to the patch-clamp setup for injection (Figure 1C). Place the membrane with attached tissue into the recording chamber perfused with warmed (37 °C) and oxygenated HEPES-buffered Ringer's solution.
4. Place the pipette containing dye against the inner limiting membrane (ILM, Figure 2A). Using micromanipulator, further advance the pipette against the ILM until it penetrates it. Inject the staining solution with a pressure 10-20 psi for 1 sec using a picospritzer. Single or multiple locations may be targeted for injection (Figure 2B). If a picospritzer is not available, a 10 ml syringe can be used to deliver a positive air pressure for injection.
5. Incubate injected tissue for 10 min. During incubation, the injected tissue can be viewed under epifluorescence illumination.
6. After labeling, the preparation may be taken to a microscope for imaging. Transfer the insert to a glass-bottom culture dish, placing retina side down (Figure 1C).
7. Add a drop of HEPES solution to keep the specimen moist.
8. Place 2-3 spots of vacuum grease around the perimeter of the plastic ring to firmly secure the insert to the bottom of the culture dish.
9. Transfer the dish to the microscope stage.

4. Paraformaldehyde and Carbodiimide Fixation

1. Fix the sample by transferring the insert into fixative for 15 min. Longer or shorter fixation times may be used, dependent on application. Both fixatives can be effectively used to fix GFP, isolectin, or fluorescent Alexa hydrazides (e.g. used for filling the cells in patch-clamp recordings).
   Note: For fixation, either 4% carbodiimide or 4% paraformaldehyde solutions may be used. To maintain adherence, fixation should be done after the tissue has already been attached to the membrane. The tissue can be fixed immediately after mounting, or after physiological recording and/or bolus injection.
2. Rinse the tissue in phosphate buffer (PB) for 30 min. After this, the tissue may be processed either as a wholemount (step 5) or cut into vertical sections and then processed (steps 6 and 7).

5. Immunostaining of Retinal Wholemounts

1. In a 24-well dish (Figure 1D) block retinal pieces on the membrane for 1 hr in a staining solution.
2. Dilute mouse anti-PSD-95 primary antibodies 1:300 in the staining solution and apply for 48 hr at room temperature in dark. Add ~100-150 μl antibody solution in each well with the tissue. To prevent drying out, fill unused wells with distilled water.
3. Rinse for 20 min in PB, 3x.
4. Incubate for 24 hr in donkey anti-mouse Alexa 568 secondary antibody dissolved 1:500 in the staining solution.
5. Rinse for 20 min in PB, 3x.
6. Place the insert with the stained tissue into a standard 3 cm Petri dish filled with PB solution. With small scissors, cut out the portion of the membrane containing the retina from the ring.
7. Transfer the retina on the membrane onto a slide retina up. Remove PB, add mounting medium. Place small pieces of a broken cover slip glass in between the slide and the coverslip glasses to avoid extensive squeezing and damage to the retina. Place coverslip.
8. Seal the coverslip in place with nail polish. Keep slides in the dark, in a refrigerator.

6. Vertical Sectioning of Membrane-mounted Tissue

1. After fixation, submerse the tissue on the membrane into the sucrose gradient solutions of 10%, 20%, and 30% sucrose for 30 min, 1 hr, and overnight, respectively. The overnight step is performed at 4 °C in refrigerator, all other steps at room temperature.
2. Using scissors, remove the membrane with the tissue from the plastic insert ring.
3. Place the cut off membrane onto Parafilm with retina up and remove as much solution as possible with Kimwipes.
4. Put the Parafilm with the tissue into the cryostat chamber and add a drop of OCT medium. Use the quick freezer device to accelerate the freezing.
5. Detach the frozen sample and flip it over.
6. Cut excess frozen OCT. Leave approximately 2 mm around the sample on three sides, and leave 5-6 mm from the side which will be attached to the holder.
7. Cover exposed retinal side (membrane side) with a drop of OCT. Leave the attaching area free.
8. Freeze the membrane with retinal tissue. Now the sample is ready for placing onto a cutting holder.
9. Place a drop of OCT onto a cold holder ring. Immediately insert the frozen sample into the drop.
10. Cut sections 10-20 μm thick, setting cryostat temperature at -19 °C. Use warm (room temperature) polysine adhesion slides. Leave the slides with the attached sections onto a heating platform for ~2-3 min (41 °C) in dark to ensure firm adhesion of sections.
11. Put the slides into a storage box and keep at -20 °C until use.

7. Immunostaining of Retinal Sections

1. Take the slides with the sections out of the freezer. Let them stay at room temperature for 1 min to remove the condensate.
2. Encircle the sections with a liquid blocker to create a barrier for further incubation.
3. Place the slides into the incubation box. Cover the bottom of the box with water to prevent evaporation of the antibody solution.
4. Rinse with PB for 5 min.
5. Prepare the same staining solutions and follow the steps as in step 5. The incubation with the primary antibody is overnight and with the secondary 1 hr. Do not use broken glass between the coverslip and the slide.

Results

Demonstrated here are two representative experiments that benefit from the use of a hydrophilized PTFE membrane to support wholemount retina during staining procedures. The first experiment, illustrates the method for fast and simple characterization of retinal vasculature, an elaborate network of blood vessels that span across multiple retinal layers (Figure 2). This approach combines bolus loading of isolectin with immersion labeling with SRH. This is an efficient method of labeling living tissue that ...

Discussion

The hydrophilized PTFE membrane's high biocompatibility and transparency in solutions is advantageous when working with living tissue. This preparation has been successfully used in earlier work for patch-clamp recordings of light responses^8-10. Here, we show how this approach can be adopted for structural analysis of the wholemount retina.

The bolus loading technique can be effectively used for labeling living tissue. This technique is aided by the structural support of the mem...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Millicell Cell Culture Insert, 12 mm, hydrophilic PTFE (Biopore), 0.4 μm	Millipore	PICM01250
Insulin syringe, 1 ml	Beckton Dickinson	309659
Scissors	Fine Science Tools	15003-08	dissection
Forceps, Dumont #55, inox	Fine Science Tools	11255-20	dissection
Cryostat	Leica	various
Confocal Microscope System	Nikon	various
Capillary glass	World Precision Instruments	1B150F-4
P-97 Flaming/Brown Micropipette Puller	Sutter Instrument Co.
Picospritzer III	Parker Hannifin
Glass Bottom Culture Dishes	MatTek Corporation	P35G-0-14-C
Petri dish	Falcon	1008
Disposable Graduated Transfer Pipettes	VWR	16001-180
Multiwell plates, 24-well	Beckton Dickinson	351147
Cover glass, #1	Electron Microscopy Sciences	72200-30
Polysine adhesion slides	Electron Microscopy Sciences	63412-01
Microscope slides	Globe	1301
Liquid Blocker	Electron Microscopy Sciences	71312
VECTASHIELD mounting medium	Vector Laboratories	H-1000
OCT medium	Sakura	4583
Parafilm laboratory film	Fisher	13-374-10
Dow Corning high vacuum grease	Sigma-Aldrich	Z273554
Mouse anti-PSD95	Millipore	MABN68	antibody
Donkey anti-mouse Alexa 568	Invitrogen	A10037	antibody
Isolectin Alexa 488	Invitrogen	I21411
Chemiblocker	Chemicon	2170S
Triton X-100	Sigma-Aldrich	T9284
Sulforhodamine	Sigma-Aldrich	341738
Carbodiimide	Thermo Scientific	22980	EDC
Paraformaldehyde	Sigma-Aldrich	P6148