Journal of Neuroscience Methods, 44 (1992) 217-223
217
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-0270/92/$05.00 NSM 01414
Photochromic intensification of diaminobenzidine reaction product in the presence of tetrazolium salts: applications for intracellular labelling and immunohistochemistry David I. Vaney Vision, Touch and Hearing Research Centre, University of Queensland, Brisbane (Australia) (Received 18 March 1992) (Revised version received 11 July 1992) (Accepted 22 July 1992)
Key words: Retina; Diaminobenzidine; Tetrazolium salts; Neurobiotin; Photochromic intensification; Immunohistochemistry The diaminobenzidine (DAB) reaction product can be greatly intensified by incubating the reacted tissue in either nitro blue tetrazolium or tetranitro blue tetrazolium and then exposing the tissue to strong light. Epi-illumination through a microscope objective enables the photochromic intensification to be carried out under direct visual control, with optimal intensification taking only 10-30 s through a 20 x objective. Alternatively, the whole preparation can be intensified in a few minutes by passing it back and forth under a fibre light guide. The method can be used to intensify cells that have been labelled either by immunoperoxidase techniques or with intracellular tracers such as horseradish peroxidase and neurobiotin.
Introduction
Diaminobenzidine (DAB) is the most widely used chromogen for the detection of peroxidase activity in immunohistochemical and intracellular labelling studies (Graham and Karnovsky, 1966). The brown DAB reaction product can be intensified by a number of methods including osmication, silver intensification (reviewed by Merchenthaler et al., 1989) and, most commonly, pre-incubation of the tissue in cobalt a n d / o r nickel ions (Adams, 1977, 1981). In this paper I report the novel finding that the DAB reaction product can be greatly intensified by incubating the re-
Correspondence: D.I. Vaney, Vision, Touch and Hearing Research Centre, Department of Physiology and Pharmacology, University of Queensland, Brisbane, Queensland 4072, Australia. Tel.: (61-7) 365-3759; FAX: (61-7) 365-1766; Email: [email protected].
acted tissue in tetrazolium salts and then exposing the tissue to strong light. This photochromic intensification method, which is very simple and very quick, greatly increases the contrast of the labelled neurones, particularly in tissue with lowbackground labelling. Thus, it is an outstanding method for intensifying neurones that have been intracellularly labelled with horseradish peroxidase or biotinylated tracers: this is illustrated for rabbit retinal neurones that were injected with neurobiotin. The method is also very effective for intensifying immunolabelled cells: this is illustrated for cat retinal neurones that showed choline acetyltransferase immunoreactivity.
Methods
Neurobiotin injection and visualization The techniques for dye filling of microscopically identified neurones in superfused rabbit
218 r e t i n a have b e e n described in detail elsewhere (Vaney, 1984, 1991; V a n e y et al., 1991). Briefly, the retina was dissected from the sclera a n d m o u n t e d on a black Millipore filter which was t h e n placed in a tissue c h a m b e r o n a fixed-stage microscope a n d s u p e r f u s e d with c a r b o g e n a t e d
A m e s m e d i u m at 1 m l / m i n . T h e retinal n e u r o n e s were stained in vitro by a d d i n g a few drops of 1 m M acridine o r a n g e to the c h a m b e r (Dacey, 1989). T h e injection micropipettes c o n t a i n e d 4% N-(2-aminoethyl)-biotinamide hydrochloride (neurobiotinXM; Vector Labs) a n d 2% lucifer yel-
.A J
Fig. 1. Micrographs of the same field of a rabbit retinal wholemount, taken before (A) and after (B) photochromic intensification in the presence of nitro blue tetrazolium. The labelled neurone marked with an arrow is an interstitial amacrine cell that was injected with neurobiotin under microscopic control: it is tracer-coupled to neighbouring interstitial cells, whose processes ramify in the middle of the inner plexiform layer. Photochromic intensification greatly increases the contrast of the DAB reaction product in the neurobiotin-filled cells. Scale bar: 50 p.m.
219 low CH (Sigma) in 0.1 M Tris buffer, pH 7.6. The lucifer yellow enabled one to visualize the micropipette tip under violet excitation (355-425 nm) and to confirm that the target cell had been penetrated. The neurobiotin was iontophoresed with a positive current of 0.5 nA for 30-120 s (Vaney, 1991). The retina was left to equilibrate in Ames medium for at least 30 min and then it was fixed for 60 min in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The tissue was removed from the filter, incubated in 0.5% Triton-X in phosphate buffer overnight, and then reacted with 1:500 streptavidin-biotinylated-peroxidase complex (Amersham) for 3 h. The retina was incubated for 10 min in 0.05% 3,3'diaminobenzidine (DAB; Sigma) in 0.1 M Tris buffer, pH 7.6, reacted with 0.01% hydrogen peroxide for 10 min, and then washed in Tris buffer.
Photomicrography
Choline acetyltransferase-immunohistochemistry
Photochromic intensification of neurobiotin-filled cells
To illustrate photochromic intensification of immunolabelled cells, a cat retina was processed for choline acetyltransferase-immunohistochemistry (Schmidt et al., 1985; Pourcho and Osman, 1986; Vaney, 1990). The cat was deeply anaesthetised with sodium pentobarbitone and perfused transcardially with carbogenated Ames medium for 15 min. The eyes were then enucleated, hemisected and the eyecups fixed for 30 min in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The retina was then dissected from the sclera and incubated for 2 days at 4°C in a rat monoclonal antibody to choline acetyltransferase (1: 10; Boehringer-Mannheim) in phosphate buffer with 5% sucrose, 2% BSA, 0.5% Triton-X and 0.1% sodium azide. The retina was then incubated with biotinylated anti-rat immunoglobulin (1:100; Amersham) and streptavidin-biotinylated-peroxidase complex (1 : 300; Amersham), both for 3 h, and then reacted with DAB and hydrogen peroxide. For heavy-metal intensification of the DAB reaction product (Adams, 1977, 1981), parts of the cat retina were incubated in either 0.01% CoCI 2 or 0.01% NiNH3SO 4 in 0.05% DAB solution, prior to reaction with the hydrogen peroxide.
Each set of micrographs, which illustrate labelled neurones before and after photochromic intensification, were produced under identical conditions. The negatives were taken using automatic exposure on a single roll of Kodak T-MAX 100 film. The micrographs were printed on Ilford Multigrade III paper at grade 3.5, with a constant exposure time for each set. The micrographs in this paper were taken with the retina cover-slipped in the tetrazolium solution. After thorough washing, however, the intensified tissue can be either cover-slipped in phosphate-buffered glycerine or mounted on a gelatinized slide, dehydrated, cleared 'and coverslipped in DePex.
Results
Neurobiotin is a biotinylated tracer which has recently been developed for intracellular labelling of neurones (Kita and Armstrong, 1991; Vaney, 1991). The molecular weight of neurobiotin (286 Da) is less than that of either biocytin (373 Da) (Horikawa and Armstrong, 1988) or lucifer yellow (457 Da) (Stewart, 1978), enabling the tracer to diffuse readily through gap junctions (Vaney, 1991). Fig. 1 shows an interstitial amacrine cell in the rabbit retina that was injected with neurobiotin in an isolated superfused preparation, some 80 min before fixation. After DAB processing, the injected neurone showed homologous coupling to about 50 other interstitial amacrine cells, 7 of which are included in the micrograph. The tracer-coupled neurones were labelled less intensely than the injected neurone, making it difficult to map their dendritic morphologies prior to photochromic intensification (Fig. 1A). Following a 5-min incubation of the retina in 0.02% nitro blue tetrazolium (NBT) in 0.1 M Tris buffer, pH 8.2, the wholemount was cover-slipped in the same solution, and select retinal fields were then illuminated with green light through a 20 x objective (NA 0.5). The photochromic intensification of the DAB reaction product could
220
be monitored microscopically: the labelled neurones began to darken within seconds, reaching maximum contrast after 10-30 s (Fig. 1B). Longer periods of illumination caused the background to darken, thus reducing the contrast of the labelled neurones to a sub-optimal level. The photochromic intensification method was particularly useful for visualizing the full extent of the axon-like processes arising from low density amacrine cells (Dacey, 1988, 1989, 1990; Vaney et al., 1988; Sagar, 1990; Vaney, 1992; Famiglietti, 1992a,b). Prior to intensification, the thin varicose axons of these neurones could only be traced for about 2 mm from the cell body whereas, following intensification, the same processes could be traced for up to 5 mm, thus establishing that such axon-bearing amacrine ceils are the largest neurones in the retina (Vaney, 1992). Moreover, the unintensified axons had to be traced under a 40 × objective whereas the intensified axons could be resolved under a 16 × objective.
A
B I !
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Photochromic intensification of immunolabelled cells Fig. 2 shows peripheral fields of a wholemounted cat retina that was processed for choline acetyltransferase-immunohistochemistry. The cell bodies of the cholinergic amacrine cells were located in either the inner nuclear layer (out of focus) or displaced to the ganglion cell layer (in focus) (Schmidt et al., 1985; Pourcho and Osman, 1986; Vaney, 1990). Prior to intensification, the DAB reaction product in the cholinergic cells
Fig. 2. Micrographs of a cat retinal wholemount processed for choline acetyltransferase-immunohistochemistry, with the focus on the displaced cholinergic amacrine cells in the ganglion cell layer. Comparison of the micrographs taken before (A) and after (B) photochromic intensification shows that the method greatly increases the contrast of the D A B reaction product in the immunoreactive cells. Nickel intensification (C) of the D A B reaction product results in labelling of intermediate contrast. Scale bar: 50/xm.
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221 appeared weak (Fig. 2A) whereas, after 10 s of photochromic intensification in 0.02% NBT, the contrast of the immunoreactive cells was greatly increased (Fig. 2B). Like other intensification techniques, photochromic intensification enables the use of higher dilutions of the antibodies and other reagents, improving not only the sensitivity but also the specificity of the immunohistochemical labelling. For comparison with other intensification methods, adjacent pieces of the immunolabelled retina were incubated with either cobalt or nickel ions during the DAB processing. Both the c o b a l t - D A B and nickel-DAB reaction products were intermediate in contrast between the unintensified reaction product and the photochromic reaction product (Fig. 2C). The photochromic method produced only moderate intensification of the n i c k e l - D A B reaction product and it was ineffective for intensifying the c o b a l t - D A B reaction product. Thus, better results were obtained when photochromic intensification was used on its own, rather than in combination with other intensification methods. Glycerine-mounted retinae, which either contained neurobiotin-filled cells or had been processed for tyrosine hydroxylase-immunohistochemistry (Vaney, 1990), were successfully intensified by the photochromic procedure following rehydration of the tissue. However, the contrast of intensified ceils in archival material was not as great as in fresh material.
Optimal parameters for photochromic intensification The optimal parameters for photochromic intensification of the DAB reaction product were assayed on rabbit retinal wholemounts that had been processed for protein kinase C - i m m u n o histochemistry to reveal the rod bipolar cells (Negishi et al., 1988; Young and Vaney, 1991). Tetrazolium salts. Most of the water-soluble tetrazolium salts in the Sigma catalogue were tested for their effectiveness. Marked intensification was obtained only with tetrazolium salts that are commonly used as substrates in enzyme histochemistry, namely nitro blue tetrazolium (NBT) and tetranitro blue tetrazolium (TNBT). With
these salts, both of which possess a low redox potential, the intensification procedure gave rise to a dark purple reaction product in the labelled cells. Photochromic intensification was effective over a wide concentration range of the tetrazolium salt and it was imperative to wash the intensified tissue overnight to remove the remaining tetrazolium salt (see Discussion). Buffer pH. Photochromic intensification was most effective when the tissue was incubated in basic buffer, with optimal results being obtained around pH 8. Wavelength. For epi-illumination, the light from a 50-W mercury lamp was passed through either a green filter (530-585 nm; Zeiss filter set 00 for Texas red excitation), a blue filter (450-490 nm; Zeiss filter set 10 for selective FITC excitation), or an ultraviolet filter (365 nm peak; Zeiss filter set 02). Photochromic intensification was most rapid under green light, although it is noted that the excitation filters were not matched for isoluminance. Under ultraviolet light, the intensification occurred more slowly and the contrast of the immunoreactive neurones was not as great. Illumination time. The time required for photochromic intensification was proportional to the area illuminated by the objective. Using a 5 x objective (NA 0.15), a field of 4 mm diameter could be intensified within 10 min. As an alternative to microscopic illumination, the cover-slipped preparation was placed under a fibre light guide (8 mm diameter) connected to a 150-W halogen lamp, and the slide moved back and forth by hand. Using this procedure, a complete retinal wholemount could be photochromically intensified in only a few minutes, with the degree of intensification being checked microscopically every 20-30 s.
Discussion
Photochromic intensification of the diaminobenzidine (DAB) reaction product was discovered by accident when I processed a neurobiotinlabelled retina for NADPH-diaphorase histochemistry but did not wash the tissue thoroughly before microscopic examination: the transmitted
222
illumination from the substage condenser was sufficient to slowly intensify the neurobiotin-filled neurones. It transpired that NBT was the only chemical in the N A D P H - d i a p h o r a s e reaction mixture that was necessary for photochromic intensification. Other experiments established that the intensification was mediated by the DAB reaction product and not by the bound peroxidase, either alone or when incubated with DAB. The actual mechanism of photochromic intensification is unknown, but it seems likely that NBT and TNBT, when excited by light, can be reduced by the DAB reaction product, forming insoluble formazan compounds (Altman, 1976). Photochromic intensification has a number of advantages over other methods of intensifying the DAB reaction product. First, photochromic intensification is very simple and can be performed under direct microscopic control on an open bench. Although silver intensification techniques are very sensitive, they are rather laborious and require extreme care (Merchenthaler et al., 1989). Osmication must be performed in a fume cupboard, thus precluding continuous monitoring of the intensification process; cobalt-nickel intensification is an all-or-none procedure, where the parameters are selected before DAB processing (Adams, 1977, 1981). Second, photochromic intensification produces more intense labelling than cobalt-nickel methods and lower background than osmication, which may darken the surface of the preparation, thus reducing the contrast of intensified cells within the tissue. On the negative side, Sigma now warns that nitro blue terazolium is a poison that may be fatal or cause blindness if swallowed. Its vapour is harmful and it cannot be made non-poisonous. Thus, the tetrazolium salts should always be weighed out and dissolved inside a fume cupboard. Gloves should be worn during solution preparation, tissue incubation, photochromic intensification and tissue washing. The tetrazolium toxicity notwithstanding, photochromic intensification promises to become a common laboratory procedure not simply because it produces outstanding re.suits, but because people like using the method.
Acknowledgements D.I.V. is an N H M R C Senior Research Fellow. I thank Dr. David Pow for useful discussions and Dr. Ian Gynther for comments on the manuscript.
References Adams, J.C. (1977) Technical considerations on the use of horseradish peroxidase as a neuronal marker. Neuroscience, 2: 141-145. Adams, J.C. (1981) Heavy metal intensification of DAB-based HRP reaction product. J. Histochem. Cytochem., 29: 775. Altman, F.P. (1976) Tetrazolium salts and formazans. Prog. Histochem. Cytochem., 9: 1-56. Dacey, D.M. (1988) Dopamine-accumulating retinal neurons revealed by in vitro fluorescence display a unique morphology. Science, 240:1196-1 ! 98. Dacey, D.M. (1989) Axon-bearing amacrine cells of the macaque monkey retina. J. Comp. Neurol., 284: 275-293. Dacey, D.M. (1990) The dopaminergic amacrine cell. J. Comp. Neurol., 301: 461-489. Famiglietti, E.V. (1992a) Polyaxonal amacrine ceils of rabbit retina: morphology and stratification of PAl cells. J. Comp. Neurol., 316: 391-405. Famiglietti, E.V. (1992b) Polyaxonal amacrine cells of rabbit retina: PA2, PA3, and PA4 cells. J. Comp. Neurol., 316: 422-446. Graham, R.C. and Karnovsky, M.J. (1966) The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem., 14:291-302 Horikawa, K. and Armstrong, W.E. (1988) A versatile means of intraeellular labeling: injection of biocytin and its detection with avidin conjugates. J. Neurosci. Methods, 25: 1-11. Kita, H. and Armstrong, W. (1991) A biotin-containing compound N-(2-aminoethyl) biotinamide for intracellular labeling and neuronal tracing studies: comparison with biocytin. J. Neurosci. Methods, 37: 141-150. Merchenthaler, I., Gallyas, F. and Liposits, Z. (1989) Silver intensification in immunocytochemistry. In: G.R. Bullock and P. Petrusz (Eds.), Techniques in Immunocytochemistry, Vol. 4, Academic Press, London, pp. 217-252. Negishi, K., Kato, S. and Teranishi, T. (1988) Dopamine cells and rod bipolar cells contain protein kinase C-like immunoreactivity in some vertebrate retinas. Neurosei. Lett.. 94: 247-252. Poureho, R.G. and Osman, K. (1986) Cytochemical identification of cholinergic amacrine cells in cat retina. J. Comp. Neurol., 247: 497-504. Sagar, S.M. (1990) NADPH-diaphorase reactive neurons of the rabbit retina: differential sensitivity to excitotoxins and unusual morphologic features. J. Comp. Neurol., 300: 309-319
223 Schmidt, M., W~issle, H. and Humphrey, M. (1985) Number and distribution of putative cholinergic neurons in the cat retina. Neurosci. Lett., 59: 235-240. Stewart, W.W. (1978) Functional connections between cells as revealed by dye-coupling with a highly fluorescent naphthalimide tracer. Cell, 14: 741-759. Vaney, D.I. (1984) Coronate amacrine cells in the rabbit retina have the starburst dendritic morphology. Proc. R. Soc. Lond. B, 220: 501-508. Vaney, D.I. (1990) The mosaic of amacrine cells in the mammalian retina. Prog. Retinal Res., 9: 49-100. Vaney, D.I. (1991) Many diverse types of retinal neurons show tracer coupling when injected with biocytin or Neurobiotin. Neurosci. Lett., 125: 187-190.
Vaney, D.I. (1992) Neurobiotin injection reveals the complete morphology and coupled somatic mosaic of axon-bearing amacrine cells in mammalian retinae. Proc. Aust. Neurosci. Soc., 3: 63. Vaney, D.I., Peichl, L. and Boycott, B.B. (1988) Neurofibrillar long-range amacrine cells in mammalian retinae. Proc. R. Soc. Lond. B, 235: 203-219. Vaney, D.I., Gynther I.C. and Young, H.M. (1991) Rod-signal interneurons in the rabbit retina: 2. AII amacrine cells. J. Comp. Neurol., 310: 154-169. Young, H.M. and Vaney, D.I. (1991) Rod-signal interneurons in the rabbit retina. 1. Rod bipolar cells. J. Comp. Neurol., 310: 139-153.
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© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-0270/92/$05.00 NSM 01414
Photochromic intensification of diaminobenzidine reaction product in the presence of tetrazolium salts: applications for intracellular labelling and immunohistochemistry David I. Vaney Vision, Touch and Hearing Research Centre, University of Queensland, Brisbane (Australia) (Received 18 March 1992) (Revised version received 11 July 1992) (Accepted 22 July 1992)
Key words: Retina; Diaminobenzidine; Tetrazolium salts; Neurobiotin; Photochromic intensification; Immunohistochemistry The diaminobenzidine (DAB) reaction product can be greatly intensified by incubating the reacted tissue in either nitro blue tetrazolium or tetranitro blue tetrazolium and then exposing the tissue to strong light. Epi-illumination through a microscope objective enables the photochromic intensification to be carried out under direct visual control, with optimal intensification taking only 10-30 s through a 20 x objective. Alternatively, the whole preparation can be intensified in a few minutes by passing it back and forth under a fibre light guide. The method can be used to intensify cells that have been labelled either by immunoperoxidase techniques or with intracellular tracers such as horseradish peroxidase and neurobiotin.
Introduction
Diaminobenzidine (DAB) is the most widely used chromogen for the detection of peroxidase activity in immunohistochemical and intracellular labelling studies (Graham and Karnovsky, 1966). The brown DAB reaction product can be intensified by a number of methods including osmication, silver intensification (reviewed by Merchenthaler et al., 1989) and, most commonly, pre-incubation of the tissue in cobalt a n d / o r nickel ions (Adams, 1977, 1981). In this paper I report the novel finding that the DAB reaction product can be greatly intensified by incubating the re-
Correspondence: D.I. Vaney, Vision, Touch and Hearing Research Centre, Department of Physiology and Pharmacology, University of Queensland, Brisbane, Queensland 4072, Australia. Tel.: (61-7) 365-3759; FAX: (61-7) 365-1766; Email: [email protected].
acted tissue in tetrazolium salts and then exposing the tissue to strong light. This photochromic intensification method, which is very simple and very quick, greatly increases the contrast of the labelled neurones, particularly in tissue with lowbackground labelling. Thus, it is an outstanding method for intensifying neurones that have been intracellularly labelled with horseradish peroxidase or biotinylated tracers: this is illustrated for rabbit retinal neurones that were injected with neurobiotin. The method is also very effective for intensifying immunolabelled cells: this is illustrated for cat retinal neurones that showed choline acetyltransferase immunoreactivity.
Methods
Neurobiotin injection and visualization The techniques for dye filling of microscopically identified neurones in superfused rabbit
218 r e t i n a have b e e n described in detail elsewhere (Vaney, 1984, 1991; V a n e y et al., 1991). Briefly, the retina was dissected from the sclera a n d m o u n t e d on a black Millipore filter which was t h e n placed in a tissue c h a m b e r o n a fixed-stage microscope a n d s u p e r f u s e d with c a r b o g e n a t e d
A m e s m e d i u m at 1 m l / m i n . T h e retinal n e u r o n e s were stained in vitro by a d d i n g a few drops of 1 m M acridine o r a n g e to the c h a m b e r (Dacey, 1989). T h e injection micropipettes c o n t a i n e d 4% N-(2-aminoethyl)-biotinamide hydrochloride (neurobiotinXM; Vector Labs) a n d 2% lucifer yel-
.A J
Fig. 1. Micrographs of the same field of a rabbit retinal wholemount, taken before (A) and after (B) photochromic intensification in the presence of nitro blue tetrazolium. The labelled neurone marked with an arrow is an interstitial amacrine cell that was injected with neurobiotin under microscopic control: it is tracer-coupled to neighbouring interstitial cells, whose processes ramify in the middle of the inner plexiform layer. Photochromic intensification greatly increases the contrast of the DAB reaction product in the neurobiotin-filled cells. Scale bar: 50 p.m.
219 low CH (Sigma) in 0.1 M Tris buffer, pH 7.6. The lucifer yellow enabled one to visualize the micropipette tip under violet excitation (355-425 nm) and to confirm that the target cell had been penetrated. The neurobiotin was iontophoresed with a positive current of 0.5 nA for 30-120 s (Vaney, 1991). The retina was left to equilibrate in Ames medium for at least 30 min and then it was fixed for 60 min in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The tissue was removed from the filter, incubated in 0.5% Triton-X in phosphate buffer overnight, and then reacted with 1:500 streptavidin-biotinylated-peroxidase complex (Amersham) for 3 h. The retina was incubated for 10 min in 0.05% 3,3'diaminobenzidine (DAB; Sigma) in 0.1 M Tris buffer, pH 7.6, reacted with 0.01% hydrogen peroxide for 10 min, and then washed in Tris buffer.
Photomicrography
Choline acetyltransferase-immunohistochemistry
Photochromic intensification of neurobiotin-filled cells
To illustrate photochromic intensification of immunolabelled cells, a cat retina was processed for choline acetyltransferase-immunohistochemistry (Schmidt et al., 1985; Pourcho and Osman, 1986; Vaney, 1990). The cat was deeply anaesthetised with sodium pentobarbitone and perfused transcardially with carbogenated Ames medium for 15 min. The eyes were then enucleated, hemisected and the eyecups fixed for 30 min in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The retina was then dissected from the sclera and incubated for 2 days at 4°C in a rat monoclonal antibody to choline acetyltransferase (1: 10; Boehringer-Mannheim) in phosphate buffer with 5% sucrose, 2% BSA, 0.5% Triton-X and 0.1% sodium azide. The retina was then incubated with biotinylated anti-rat immunoglobulin (1:100; Amersham) and streptavidin-biotinylated-peroxidase complex (1 : 300; Amersham), both for 3 h, and then reacted with DAB and hydrogen peroxide. For heavy-metal intensification of the DAB reaction product (Adams, 1977, 1981), parts of the cat retina were incubated in either 0.01% CoCI 2 or 0.01% NiNH3SO 4 in 0.05% DAB solution, prior to reaction with the hydrogen peroxide.
Each set of micrographs, which illustrate labelled neurones before and after photochromic intensification, were produced under identical conditions. The negatives were taken using automatic exposure on a single roll of Kodak T-MAX 100 film. The micrographs were printed on Ilford Multigrade III paper at grade 3.5, with a constant exposure time for each set. The micrographs in this paper were taken with the retina cover-slipped in the tetrazolium solution. After thorough washing, however, the intensified tissue can be either cover-slipped in phosphate-buffered glycerine or mounted on a gelatinized slide, dehydrated, cleared 'and coverslipped in DePex.
Results
Neurobiotin is a biotinylated tracer which has recently been developed for intracellular labelling of neurones (Kita and Armstrong, 1991; Vaney, 1991). The molecular weight of neurobiotin (286 Da) is less than that of either biocytin (373 Da) (Horikawa and Armstrong, 1988) or lucifer yellow (457 Da) (Stewart, 1978), enabling the tracer to diffuse readily through gap junctions (Vaney, 1991). Fig. 1 shows an interstitial amacrine cell in the rabbit retina that was injected with neurobiotin in an isolated superfused preparation, some 80 min before fixation. After DAB processing, the injected neurone showed homologous coupling to about 50 other interstitial amacrine cells, 7 of which are included in the micrograph. The tracer-coupled neurones were labelled less intensely than the injected neurone, making it difficult to map their dendritic morphologies prior to photochromic intensification (Fig. 1A). Following a 5-min incubation of the retina in 0.02% nitro blue tetrazolium (NBT) in 0.1 M Tris buffer, pH 8.2, the wholemount was cover-slipped in the same solution, and select retinal fields were then illuminated with green light through a 20 x objective (NA 0.5). The photochromic intensification of the DAB reaction product could
220
be monitored microscopically: the labelled neurones began to darken within seconds, reaching maximum contrast after 10-30 s (Fig. 1B). Longer periods of illumination caused the background to darken, thus reducing the contrast of the labelled neurones to a sub-optimal level. The photochromic intensification method was particularly useful for visualizing the full extent of the axon-like processes arising from low density amacrine cells (Dacey, 1988, 1989, 1990; Vaney et al., 1988; Sagar, 1990; Vaney, 1992; Famiglietti, 1992a,b). Prior to intensification, the thin varicose axons of these neurones could only be traced for about 2 mm from the cell body whereas, following intensification, the same processes could be traced for up to 5 mm, thus establishing that such axon-bearing amacrine ceils are the largest neurones in the retina (Vaney, 1992). Moreover, the unintensified axons had to be traced under a 40 × objective whereas the intensified axons could be resolved under a 16 × objective.
A
B I !
h
Photochromic intensification of immunolabelled cells Fig. 2 shows peripheral fields of a wholemounted cat retina that was processed for choline acetyltransferase-immunohistochemistry. The cell bodies of the cholinergic amacrine cells were located in either the inner nuclear layer (out of focus) or displaced to the ganglion cell layer (in focus) (Schmidt et al., 1985; Pourcho and Osman, 1986; Vaney, 1990). Prior to intensification, the DAB reaction product in the cholinergic cells
Fig. 2. Micrographs of a cat retinal wholemount processed for choline acetyltransferase-immunohistochemistry, with the focus on the displaced cholinergic amacrine cells in the ganglion cell layer. Comparison of the micrographs taken before (A) and after (B) photochromic intensification shows that the method greatly increases the contrast of the D A B reaction product in the immunoreactive cells. Nickel intensification (C) of the D A B reaction product results in labelling of intermediate contrast. Scale bar: 50/xm.
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221 appeared weak (Fig. 2A) whereas, after 10 s of photochromic intensification in 0.02% NBT, the contrast of the immunoreactive cells was greatly increased (Fig. 2B). Like other intensification techniques, photochromic intensification enables the use of higher dilutions of the antibodies and other reagents, improving not only the sensitivity but also the specificity of the immunohistochemical labelling. For comparison with other intensification methods, adjacent pieces of the immunolabelled retina were incubated with either cobalt or nickel ions during the DAB processing. Both the c o b a l t - D A B and nickel-DAB reaction products were intermediate in contrast between the unintensified reaction product and the photochromic reaction product (Fig. 2C). The photochromic method produced only moderate intensification of the n i c k e l - D A B reaction product and it was ineffective for intensifying the c o b a l t - D A B reaction product. Thus, better results were obtained when photochromic intensification was used on its own, rather than in combination with other intensification methods. Glycerine-mounted retinae, which either contained neurobiotin-filled cells or had been processed for tyrosine hydroxylase-immunohistochemistry (Vaney, 1990), were successfully intensified by the photochromic procedure following rehydration of the tissue. However, the contrast of intensified ceils in archival material was not as great as in fresh material.
Optimal parameters for photochromic intensification The optimal parameters for photochromic intensification of the DAB reaction product were assayed on rabbit retinal wholemounts that had been processed for protein kinase C - i m m u n o histochemistry to reveal the rod bipolar cells (Negishi et al., 1988; Young and Vaney, 1991). Tetrazolium salts. Most of the water-soluble tetrazolium salts in the Sigma catalogue were tested for their effectiveness. Marked intensification was obtained only with tetrazolium salts that are commonly used as substrates in enzyme histochemistry, namely nitro blue tetrazolium (NBT) and tetranitro blue tetrazolium (TNBT). With
these salts, both of which possess a low redox potential, the intensification procedure gave rise to a dark purple reaction product in the labelled cells. Photochromic intensification was effective over a wide concentration range of the tetrazolium salt and it was imperative to wash the intensified tissue overnight to remove the remaining tetrazolium salt (see Discussion). Buffer pH. Photochromic intensification was most effective when the tissue was incubated in basic buffer, with optimal results being obtained around pH 8. Wavelength. For epi-illumination, the light from a 50-W mercury lamp was passed through either a green filter (530-585 nm; Zeiss filter set 00 for Texas red excitation), a blue filter (450-490 nm; Zeiss filter set 10 for selective FITC excitation), or an ultraviolet filter (365 nm peak; Zeiss filter set 02). Photochromic intensification was most rapid under green light, although it is noted that the excitation filters were not matched for isoluminance. Under ultraviolet light, the intensification occurred more slowly and the contrast of the immunoreactive neurones was not as great. Illumination time. The time required for photochromic intensification was proportional to the area illuminated by the objective. Using a 5 x objective (NA 0.15), a field of 4 mm diameter could be intensified within 10 min. As an alternative to microscopic illumination, the cover-slipped preparation was placed under a fibre light guide (8 mm diameter) connected to a 150-W halogen lamp, and the slide moved back and forth by hand. Using this procedure, a complete retinal wholemount could be photochromically intensified in only a few minutes, with the degree of intensification being checked microscopically every 20-30 s.
Discussion
Photochromic intensification of the diaminobenzidine (DAB) reaction product was discovered by accident when I processed a neurobiotinlabelled retina for NADPH-diaphorase histochemistry but did not wash the tissue thoroughly before microscopic examination: the transmitted
222
illumination from the substage condenser was sufficient to slowly intensify the neurobiotin-filled neurones. It transpired that NBT was the only chemical in the N A D P H - d i a p h o r a s e reaction mixture that was necessary for photochromic intensification. Other experiments established that the intensification was mediated by the DAB reaction product and not by the bound peroxidase, either alone or when incubated with DAB. The actual mechanism of photochromic intensification is unknown, but it seems likely that NBT and TNBT, when excited by light, can be reduced by the DAB reaction product, forming insoluble formazan compounds (Altman, 1976). Photochromic intensification has a number of advantages over other methods of intensifying the DAB reaction product. First, photochromic intensification is very simple and can be performed under direct microscopic control on an open bench. Although silver intensification techniques are very sensitive, they are rather laborious and require extreme care (Merchenthaler et al., 1989). Osmication must be performed in a fume cupboard, thus precluding continuous monitoring of the intensification process; cobalt-nickel intensification is an all-or-none procedure, where the parameters are selected before DAB processing (Adams, 1977, 1981). Second, photochromic intensification produces more intense labelling than cobalt-nickel methods and lower background than osmication, which may darken the surface of the preparation, thus reducing the contrast of intensified cells within the tissue. On the negative side, Sigma now warns that nitro blue terazolium is a poison that may be fatal or cause blindness if swallowed. Its vapour is harmful and it cannot be made non-poisonous. Thus, the tetrazolium salts should always be weighed out and dissolved inside a fume cupboard. Gloves should be worn during solution preparation, tissue incubation, photochromic intensification and tissue washing. The tetrazolium toxicity notwithstanding, photochromic intensification promises to become a common laboratory procedure not simply because it produces outstanding re.suits, but because people like using the method.
Acknowledgements D.I.V. is an N H M R C Senior Research Fellow. I thank Dr. David Pow for useful discussions and Dr. Ian Gynther for comments on the manuscript.
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