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Optimal Methods for Preparation and Immunostaining of Iris, Ciliary Body, and Choroidal Wholemounts

From the Department of Anatomy and Human Biology, The University of Western Australia, Nedlands, Perth.

	Abstract

Top Abstract Introduction Methods Discussion Appendix 1 References

 
PURPOSE. Investigations into the biology of resident and infiltrating immune cells in the uveal tract of the rodent eye have been greatly aided by the use of tissue wholemount methods. These methods offer a number of advantages over conventional histological and frozen section techniques. The purpose of this article is to provide a detailed step by step guide to aid others who may wish to use this METHOD. METHODS. A detailed description of whole-body perfusion fixation, dissection and isolation of the iris-ciliary body from the anterior segment and the choroid from the posterior segment is provided. In addition, the techniques used to handle whole tissue pieces during single and double immunohistochemical staining protocols, as well as the staining protocols themselves, are DESCRIBED. RESULTS. In refining the techniques described, the author has catalogued a number of frequent problems which compromise immunohistochemical staining results. A troubleshooting guide aimed to help identify the cause of common problems and with some suggested remedies is PROVIDED. CONCLUSIONS. Although tissue wholemounts are frequently used in retinal research, a similar approach to investigating the components of the uveal tract has only recently been applied. The methods described in this article will provide sufficient detail for other investigators to obtain maximum benefit from this alternative approach and provide an additional technique to assist in their investigations of ocular immunobiology.

	Introduction

Top Abstract Introduction Methods Discussion Appendix 1 References

 
Investigations in the past few years in the author’s laboratory have focused on the biology of various types of immune cells in the normal and inflamed rodent uveal tract.^{1 2 3 4 5 6} These studies have been greatly advanced by the use of tissue wholemounts combined with immunohistochemical or histochemical methods. These techniques are routinely used in preference to conventional methods, such as wax histology or frozen tissue sections. The advantages of this approach include economy of tissue preparation time, visualization of the morphology of entire cells and cell networks, and the increased sampling of tissue. The value of this technical approach relies heavily on adequate preservation of cell morphology, optimal antigen preservation, low background staining, minimization of tissue damage during dissection and handling, and careful mounting of the specimens for photography and examination. Uveal tract wholemount preparations are most informative and rewarding when applied to albino animals in which uveal tissues are sufficiently thin and transparent to allow transillumination and resolution of individual stained cells. Thus, albino rats and mice are the most commonly chosen experimental animals. Pigmented animal strains can be studied but require bleaching to allow conventional microscopic examination. Alternatively, immunofluorescence methods can be used, although problems of autofluorescence must be borne in mind.

The purpose of this article is to provide a detailed methodologic account to aid investigators who may be considering using similar approaches. It covers all aspects of tissue preparation, from whole body perfusion, microdissection, and tissue handling, to a step-by-step guide to immunohistochemical staining. In addition, there is a supporting Internet Web site with video sequences of key procedures.

	Methods

Top Abstract Introduction Methods Discussion Appendix 1 References

 
Whole-Body Perfusion
Intracardiac perfusion can be performed by using a constant-flow pump or by gravity flow. The latter method is used in the author’s laboratory. All procedures conform to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Animals can be deeply anesthetized using a variety of methods. We use an intraperitoneal injection of pentobarbitone sodium (100 mg/kg body weight; Rhone Merieux Australia, Queensland) diluted in cold phosphate-buffered saline (PBS). When the animal is adequately anesthetized (fails to respond to a foot-pinch test) it is pinned by the feet supine to a corkboard situated inside a laminar flow cabinet. The fur on the chest and abdomen is dampened with 70% ethanol to prevent hair from contaminating wet tissue specimens. An abdominal incision is made in the midsagittal plane from the symphysis pubis to the xiphoid process. The cut is continued rostrally as paired parasagittal incisions in the chest wall, thus avoiding the paired internal thoracic arteries that lie close to the sternum. The sternum is then elevated and pinned rostrally to allow visualization of the chest cavity. Thymic tissue is displaced to expose the ascending aorta.

The apex of the heart is grasped gently with artery forceps, and a 2-mm partial-thickness scalpel incision is made in the left ventricle. A blunted wide-bore cannula (2–3-mm internal diameter) is inserted through the incision and gently pushed rostrally until the tip is seen within the lumen of the ascending aorta. The cannula is connected by wide-bore clear plastic tubing to a three-way valve. Reservoirs of cold heparinized PBS (1 IU heparin per milliliter PBS) and cold fresh 2% or 4% paraformaldehyde are connected to the other ends of the three-way valve. These reservoirs can be elevated to varying heights by using a pulley mechanism. Elevation to approximately 1200 mm above the height of the animal ensures adequate perfusion pressure. After cannulation of the heart, the valve to the PBS reservoir is opened, and a 1- to 2-mm cut is immediately made in the wall of the right atrium to allow blood and perfusate to escape. The volume of perfusate required per animal is approximately 60 to 80 ml for mice and 250 to 350 ml for rats. After the initial PBS perfusion (approximately 5–10 minutes), virtually no trace of blood should be evident leaving the heart. The three-way valve is changed to allow fixative to enter the heart. Within 5 to 10 seconds of changing to fixative, evidence of muscular spasms should be apparent. A volume of fixative equal to that recommended for PBS should ensure satisfactory fixative perfusion. Signs of adequate perfusion include movements in the limbs and tail (indicating fixative has reached the extremities), clearing of all mesenteric vessels, blanching of the liver, and absence of pink tones in the tongue and eyes. Signs that the perfusion pressure or rate is too high include fixative flowing from the nostrils and expansion of subcutaneous tissues in the head and neck due to fluid accumulation. The animal should be rigid if the perfusion fixation has been successful.

After completion of the perfusion, the cannula is removed from the heart, and the eyes are enucleated, by using blunt curved enucleation scissors. Other tissues may also be dissected at this time. Useful control tissues suitable for preparation as wholemounts include the non–fat-bearing region of the mesentery of the small intestine, epidermal sheets, meninges, or other thin tissues that are suitable to be flatmounted and transilluminated for examination by light microscopy.

Microdissection of Globes and Removal of the Iris, Ciliary Body, and Choroid
Short video sequences illustrating the following steps are available (http://iaaf.anhb.uwa.edu.au/webdev/Paul/default.htm).

Dissection of eyes is best performed under a stereoscopic dissecting microscope with a fiber-optic light source directed obliquely. This serves to reduce glare and reflections from metal dissection instruments. Dissection of albino tissues is best performed on a dark, compliant cutting surface (Fig. 1.1) . Extensive experimentation has resulted in the choice of heavy-duty, black, hard rubber blocks (approximately 10 x 6 cm and 1 cm in thickness). The globe is carefully incised (1-mm cut) immediately behind the limbus, by using a sharp razor blade (Fig. 1.2) . Curved iris scissors are then used to continue the incision around the globe and separate the eye into anterior and posterior segments (Fig. 1.3) . Jeweller’s forceps are used to hold the eyecup carefully while the lens is gently teased from the anterior segment (Fig. 1.4) . Three or four radial incisions are made with the razor blade through the anterior segment to produce pie-shaped wedges consisting of cornea–sclera externally and iris–ciliary body or choroid–retina internally (Fig. 1.5) . Excess sclera, choroid, and retina posterior to the ciliary body are carefully removed with the razor blade, using downward cutting motions (not sawing actions). Gripping the central cornea with fine forceps, a beaver blade (Tooke’s knife) is carefully placed between the inner surface of the cornea and the iris. The blade is gently pushed posteriorly into the iridocorneal angle, thus breaking any remaining adhesions between the iris–ciliary body and cornea–sclera. The isolated pieces of iris–ciliary body (Fig. 1.6) are gently lifted with either a fine artist’s paintbrush or the beaver blade and transferred into clean glass vials containing fresh PBS.

View larger version (103K): [in this window] [in a new window]   Figure 1. (1) Intact enucleated albino rat eye as seen in the binocular microscope during the dissection procedure. (2) Rat eye, illustrating the position of the initial equatorial incision behind the limbus (arrows). (3) Same eye, after completion of the equatorial cut, is separated into anterior and posterior segments. Y-shaped sutures are visible on the lens. (4) Anterior segment after removal of the lens as seen when viewed from behind. (5) The anterior segment after it has been divided into three pie-shaped pieces. (6) One segment of isolated iris–ciliary body after its removal from the cornea–sclera. Note there are no blood-filled vessels visible. This acts as evidence of a successful perfusion. (7) Frontal view of the intact posterior segment. (8) An isolated portion (approximately one third) of the posterior segment that has had the neural retina (NR) removed (left) from the rest of the globe (right) which consists of the RPE (not visible in this albino eye), choroid, and sclera. (9) Thin delicate choroid (C) (left) after its separation from the inner surface of the sclera (S) (right).

It should be noted that all dissections are performed in a fluid environment (PBS or fixative). At no stage should the tissues be allowed to dry, because it causes them to shrivel and collapse, which makes it difficult to achieve evenly distributed immunostaining and good flatmounting. Furthermore, note that the irides or choroids are never actually grasped with forceps or directly handled during the dissection procedure. The first point of direct contact with these tissues is when they are lifted with the fine paintbrush or beaver blade and transferred into PBS.

Protocol for Immunohistochemical Staining of Iris and Choroidal Tissue Wholemounts
Single Immunostaining of Tissue Wholemounts.
Tissue pieces are incubated in small sealed glass vials containing prewarmed 20 mM EDTA tetrasodium for 30 minutes at 37°C. The tissue in each vial is then quickly rinsed in PBS three times at 10 minutes per rinse. Increased permeabilization of the tissue is achieved by replacing PBS in the vials with a 0.1% solution of Tween-20 (or similar detergent) in PBS plus 1% bovine serum albumin (BSA; wt/vol; 20 minutes at room temperature [RT], 22°C).

Tissue pieces are allocated to individual wells in a flat-bottomed 24-multiwell plate (Iwaki Glass, Tokyo, Japan) corresponding to the number of primary antibodies to be used in the experiment. Incubation of tissue with primary antibodies (e.g., mouse anti-rat monoclonal) is carried out at 4°C (overnight incubation) or at RT for 1 hour. All antibodies are diluted in PBS containing 1% BSA; 150 to 200 µl is placed in each well. Between each incubation, samples are thoroughly washed (three changes of PBS, 5–10 minutes each). Solutions are carefully removed from the wells and replaced using disposable plastic or glass pipettes. It is useful to place the 24-multiwell plate on a dark background to aid in visualization of the tissue pieces in the individual wells.

A directly conjugated secondary antibody (e.g., sheep anti-mouse horseradish peroxidase [HRP ]) or a biotinylated secondary antibody is applied for 40 minutes (RT). The secondary antibodies are also diluted in PBS containing 1% BSA with the addition of 10% vol/vol normal rat serum (if species of tissue under study is rat). If biotinylated secondary antibodies are chosen, this is followed by streptavidin conjugate (e.g., streptavidin-HRP).

Chromogen development is performed according to individual protocols ^{(see Appendix)}.

Development of horseradish peroxidase as a red reaction product with 3-amino-9-ethylcarbazole (AEC; Sigma, St. Louis, MO) takes place for approximately 10 minutes at 37°C in a concentration of 0.2 mg AEC per milliliter of 5 mM prewarmed acetate buffer ^(Appendix). Visualization of labeled cells as a brown reaction product can be achieved by the use of 3,3'-diaminobenzidine (DAB). Incubation is performed for 10 to 15 minutes at RT in a concentration of 12 mg DAB per milliliter PBS . A blue reaction product can be obtained by using alkaline phosphatase–conjugated secondary antibodies and visualization of labeled cells using 0.25 mg fast blue BB base (Sigma; F-0125) per milliliter of prewarmed Tris buffer and 0.125 mg naphthol AS-MX phosphate (Sigma; N 5000) per milliliter, for approximately 20 minutes at 37°C . Reactions are stopped by replacing substrate solutions with several changes of PBS and a final rinse in distilled water.

A range of chromogens can be selected, depending on the color of reaction product desired. With albino tissues, a range such as DAB (brown), AEC (red), or alkaline phosphatase-fast blue (blue) may be chosen. However, with pigmented tissues, brown and red chromogens are not recommended. Pigment (melanin) can be removed by bleaching before histochemical or immunohistochemical staining, but appropriate controls should be performed to ensure the bleaching process does not compromise antigen preservation in the tissue. Immunofluorescence methods may also be chosen when pigmented tissues are investigated; however, care should be taken that sufficient transillumination is still possible and that tissue autofluorescence is evaluated in negative controls.

Mounting of Tissue Pieces.
When chromogen development with AEC or fast blue BB has been performed, tissues are placed on subbed (chrome alum and gelatin) microscope slides in a drop of water-based mounting medium (ImmunoMount; Shandon, Pittsburgh, PA). Solvent-based mounting media destroys AEC and fast blue staining and can lead the examiner to suspect negative results. If DAB is chosen as a chromogen, tissues are placed in clean glass vials and dehydrated through graded alcohols and xylene before they are placed in a drop of mounting medium Distrene-80 Plasticizer and Xylene (DPX; BDH, Poole, England) on a microscope slide. Tissues are gently spread flat using fine paint brushes before the coverslips are applied. Small brass or lead weights are placed on the coverslips overnight to flatten samples. When tissues are mounted with water-based mounting medium, the edges of the coverslips are sealed with fingernail polish to prevent drying.

Double Immunostaining of Wholemount Tissue.
The protocol for double immunohistochemical staining is based on the work of Claassen et al.⁷ and is identical with that described, until the step subsequent to incubation in the directly conjugated secondary antibody (recognizing primary antibody 1). Tissues are washed (three times in PBS, 5 minutes each) before incubation for 3 hours at RT with a biotinylated second primary antibody (antibody 2), diluted in PBS containing 1% BSA and 10% normal mouse serum (if the primary antibody is of mouse origin). Tissues are then incubated for 40 minutes at RT in streptavidin-alkaline phosphatase diluted in 1% BSA in PBS. The alkaline phosphatase chromogen (associated with primary antibody 2) is developed first, using fast blue BB for 20 minutes at 37°C ^(Appendix). Samples are then washed thoroughly, and a second chromogen is used to visualize the HRP–conjugated secondary antibody bound to primary antibody 1. The author routinely uses AEC (10 minutes, 37°C) for this chromogen, because the red reaction product contrasts well with the blue chromogen. Double-labeled cells appear purple or a mixture of red and blue if the primary antibodies are localized to different cell compartments. A final wash in PBS and distilled water (5 minutes each) is followed by application of coverslips with water-based medium (ImmunoMount; Shandon), as has been described.

As with most techniques involving multiple steps, there are a number of stages at which problems can occur that will compromise the final results. To identify the likely cause of problems, a troubleshooting guide is included in Table 1 .

	Discussion

Top Abstract Introduction Methods Discussion Appendix 1 References

Recently, several investigators have begun to explore iris wholemount methods in a wide variety of studies,^{8 9 10} and many more have expressed an interest in using the techniques but have experienced a range of technical difficulties. It is hoped the methods described in this article provide sufficient detail for investigators to obtain maximum benefit from this alternative approach and therefore enhance the range of techniques available in their investigations of ocular immunobiology.

	Appendix 1

Top Abstract Introduction Methods Discussion Appendix 1 References

 
Substrates for Chromogen Development
A. Visualization of HRP Using AEC

1. Make a 5-mM acetate buffer (0.41 g Na acetate in 100 ml distilled water [DW]). Adjust pH to 5.0 with glacial acetic acid.
   
2. Warm 19 ml of this buffer to 37°C.
   
3. Dissolve 4 mg AEC in 0.5 ml N,N-dimethyl formamide. Add acetate buffer while stirring.
   
4. Add 7 µl of 30% H₂O₂ solution (stored in a refrigerator in a light-tight container that is kept tightly closed). The resultant solution can be filtered through a Millipore filter (Bedford, MA). If filtered, discard the first 1 ml.
   
5. Fill wells of microtiter plate with approximately 0.5 ml of this solution. Incubate at 37°C for 10 minutes.
   
6. Stop reaction by rinsing in several changes of PBS.
   
7. Rinse once in DW and mount in water-based mounting medium (author uses Immunomount; Shandon).
   

B. Visualization of HRP Using DAB

1. Thaw 1 ml aliquot of DAB solution (12 mg DDB per milliliter PBS) and add 9 ml PBS. Just before use, add 5.5 µl H₂O₂.
   
2. Place DAB solution in syringe fitted with a filter (Millipore; Bedford, MA). Note: Test DAB solution on HRP containing secondary antibody or streptavidin-HRP. A dark brown stain should appear immediately if the stain is working.
   
3. Fill wells with approximately 0.5 ml of chromogen. Incubate at RT for 10 to 15 minutes.
   
4. Wash in PBS to stop reaction.
   

C. Visualization of Alkaline Phosphatase Using Naphthol AS MX Phosphate and Fast Blue BB

1. Make Tris buffer (1.21 g Tris in 100 ml DW). Adjust pH to 8.5 with concentrated HCl.
   
2. Warm 40 ml to 37°C.
   
3. Dissolve 5 mg naphthol AS-MX phospate⁽¹⁾ in 250 µl N,N-dimethyl formamide. Add to warm Tris buffer while stirring.
   
4. Add 250 µl 2M HCl (0.86 ml concentrated HCl in 5 ml DW) to 10 mg fast blue BB⁽²⁾ , swirl, and add 250 µl fresh 4% sodium nitrite⁽³⁾ solution. (0.4 g/10 ml DW). Mix, uncapped 1 to 2 minutes and add by drops to Tris buffer on stirrer.
   
5. Dissolve 10 mg levamisole in 2 ml Tris buffer⁽⁴⁾ and add to warm Tris solution.
   
6. Ensure mixture is at 37°C and filter through 0.2/0.45 µm filter (Millipore) directly onto tissues.
   
7. Incubate in substrate solution for 20 minutes at 37°C.
   
8. Stop reaction by replacing substrate solution with 2 to 3 changes of PBS.
   

Items designated with superiors (1) through (4) can be weighed out in aliquots in advance.

	Acknowledgements

 
The author thanks the numerous students and research assistants who have assisted over several years in refining the methods described in this article.

	Footnotes

 
Supported by the National Health and Medical Research Council.

Submitted for publication September 15, 1999; revised January 18, 2000; accepted February 2, 2000.

Commercial relationships policy: N.

Corresponding author: Paul G. McMenamin, Department of Anatomy and Human Biology, The University of Western Australia, Nedlands (Perth), 6907, Western Australia. mcmenamin{at}anhb.uwa.edu.au

	References

Top Abstract Introduction Methods Discussion Appendix 1 References

 

1. McMenamin, PG, Holthouse, I, Holt, PG (1992) Class II MHC (Ia) antigen-bearing dendritic cells within the iris and ciliary body of the rat eye: distribution, phenotype, and relation to retinal microglia Immunology 77,385-393[Medline][Order article via Infotrieve]
2. McMenamin, PG, Crewe, JM, Morrison, S, Holt, PG (1994) Immunomorphologic studies of macrophages and MHC class II-positive dendritic cells in the iris and ciliary body of the rat, mouse and human eye Invest Ophthalmol Vis Sci 35,3234-3250[Abstract/Free Full Text]
3. McMenamin, PG, Crewe, J. (1995) Endotoxin-induced uveitis: kinetics and phenotype of the inflammatory cell infiltrate and the response of the resident tissue macrophages and dendritic cells in the iris and ciliary body Invest Ophthalmol Vis Sci 36,1949-1959[Abstract/Free Full Text]
4. Butler, TL, McMenamin, PG (1996) Resident and infiltrating immune cells in the uveal tract in the early and late stages of experimental autoimmune uveoretinitis Invest Ophthalmol Vis Sci 37,2195-2210[Abstract/Free Full Text]
5. McMenamin, PG (1999) Dendritic cells and macrophages in the uveal tract of the normal mouse eye Br J Ophthalmol 83,598-604[Abstract/Free Full Text]
6. McMenamin, PG, Crewe, J, Kijlstra, A. (1997) Resident and infiltrating cells in the rat iris during the early stages of experimental melanin protein-induced uveitis (EMIU) Ocul Immunol Inflamm 5,223-233[Medline][Order article via Infotrieve]
7. Claassen, E, Alder, LT, Adler, FL (1986) Double immunocytochemical staining for in situ study of allotype distribution during an anti-TNP immune response in chimeric rabbits J Histochem Cytochem 34,989-994[Abstract]
8. Pouvreau, I, Zech, J-C, Thillaye–Goldberg, B, Naud, M-C, van Rooijen, N, de Kozak, Y. (1998) Effect of macrophage depletion by liposomes containing dichloromethylene-diphosphonate on endotoxin-induced uveitis J Neuroimmunol 86,171-181[Medline][Order article via Infotrieve]
9. Yang, P, de Vos, A, Kijlstra, A. (1998) Interferon gamma immunoreactivity in iris nerve fibres during endotoxin induced uveitis in the rat Br J Ophthalmol 82,695-699[Abstract/Free Full Text]
10. Coupland, SE, Krause, L, Hoffmann, F. (1996) The influence of penetrating keratoplasty and cyclosporin A therapy on MHC class II (Ia)-positive cells in the rat iris and choroid Graefes Arch Clin Exp Ophthalmol 234,116-124[Medline][Order article via Infotrieve]

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