Use of peroxidase substrate Vector VIP® for multiple staining in light microscopy

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Abstract

The study of the distribution of a fiber input to a particular brain area and the visualization of the anatomical relationships of that input with both projection- and interneurons, requires a triple-staining that allows the unequivocal distinction of each of the three components in one and the same histological section. In this regard, we investigated the properties of a recently introduced peroxidase chromogen, VIP® (V-VIP; Vector Labs) in combination with two traditional substrates, standard diaminobenzidine (DAB, brown precipitate) and nickel-enhanced DAB (DAB-Ni, black). In rats, the anterograde tracer biotinylated dextran amine (BDA) and the retrograde tracer fluorogold (FG) were injected in the perirhinal cortex and hippocampus, respectively. Transported BDA was detected with an avidin-biotin-peroxidase complex, whereas the transported FG was detected via a PAP method. Tracing with BDA and FG was combined with parvalbumin- or calbindin-immunocytochemistry. We compared various combinations and staining sequences. The best results were obtained with a staining sequence comprising first the BDA stain with DAB-Ni as chromogen, second the FG protocol with the chromogen DAB and finally, parvalbumin- or calbinding-immunocytochemistry using the chromogen V-VIP. The order with which the chromogens were applied appeared to be critical. Partial or even total loss of V-VIP reaction product has been observed after standard dehydration in ethanol. As an alternative, a quick dehydration procedure in toluene yields much better staining. Colour separation is excellent and the sensitivity is high. This procedure may also be used for detection of any other combination of three different labels, taking the usual care to avoid cross-reactivity between antibodies.

Introduction

Knowledge of the relationships among different fiber systems and projection and/or interneurons in a particular brain region is an important goal in neuromorphology research. As part of our ongoing efforts to elucidate the anatomical organization of the cortico-hippocampal system in the rat, we are interesed in the postsynaptic target structures of efferent projections from the perirhinal cortex to the entorhinal cortex. Our special interest is to elucidate whether perirhinal efferent axons contact principal cells in the superficial layers of the entorhinal cortex, which in turn project to the hippocampus and/or presumed inhibitory entorhinal neurons (for an overall review of the neuronal circuits of interest see Witter et al., 1989; Amaral and Witter, 1995). Accordingly, we needed to develop a method for the simultaneous visualization, in one and the same section, of each of these three components. Until today, apart from dual immunofluorescence-based methods (Wessendorf, 1990), a large number of dual-staining methods for detecting two different antigens have been introduced (for a review, see Groenewegen and Wouterlood, 1990; Wouterlood and Groenewegen, 1991), mainly based on a sequential two-colour immunocytochemical paradigm (Hancock, 1986). According to this paradigm, one antigen is detected first with nickel-enhanced diaminobenzidine (DAB-Ni) and then the other using regular diaminobenzidine (DAB) as a chromogen.

Vector Labs has recently introduced several new peroxidase substrates for both light and electron microscopy. These new chromogens include the peroxidase substrate Vector™ VIP® (V-VIP), which results in a light purple reaction product which is perfectly distinguishable from the black or brown reaction product obtained with DAB-Ni or DAB, respectively. Initial results combining V-VIP and DAB-Ni or DAB look very promising for both light and electron microscopy (Zhou and Grofova, 1995).

In the present study we have chosen to visualize the perirhinal fibers with the use of the anterograde tracer biotinylated dextran amine (BDA). Entorhinal principal neurons in layers II and III were labelled via retrograde tracing with fluorogold (FG), and populations of entorhinal interneurons were stained with immunocytochemical procedures, using antiserum against parvalbumin (Pv) or calbindin (Cb). These labels were visualized in one and the same histological section with a triple staining procedure. This sensitive protocol allows to establish whether a projection targets one out of two types of neurons, or both. Moreover, it allows for the analysis of the relationships between the two neuronal populations in a particular brain structure. It is expected that a similar protocol can be used for any neuroanatomical procedure where three distinguishable chromogens are required. A preliminary report was published in an abstract form (Lanciego et al., 1996).

Section snippets

Materials and methods

Female wistar rats (n=5; body weight 200–230 g) were deeply anaesthetized (0.1 ml/100 g body weight) with an intramuscularly injected mixture of 4:3 parts of Ketaset® (1% solution of ketamine, Aesco, Boxtel, The Netherlands) and Rompun® (2% solution of xylazine, Bayer, Leverkusen, Germany) respectively. The rats were then placed in a stereotaxic frame. In a single surgical session, 5% solution of biotinylated dextran amine (BDA; Molecular Probes, Eugene, OR) in 0.01 M phosphate buffer, pH 7.25

Double-staining procedures

Two different dual-staining sequences were used to evaluate the usefulness of the V-VIP substrate as a substitute for DAB. One series of sections from each rat was used for BDA staining with DAB-Ni and FG with DAB, while in the other series BDA was stained with DAB-Ni and FG with V-VIP. In all cases, the V-VIP reaction product showed a distribution identical to that obtained when DAB was used. The appearance of the FG injection sites was similar, regardless of the chromogen employed. The same

Discussion

The sequential triple staining procedure described here, combining the peroxidase substrates DAB-Ni, DAB and V-VIP, resulted in a sensitive three-colour paradigm that allows unequivocal detection of three different labels. The two tracers used in the present study have been widely used and various application procedures as well as uptake and transport characteristics have already been reported in detail (Schmued and Heimer, 1990; Brandt and Apkarian, 1992; Veenman et al., 1992; Rajakumar et

Acknowledgements

We thank Dr Theo van Haeften for his stimulating discussion and suggestions that helped us to improve the manuscript. Dirk de Jong, Barbara Jorritsma-Byham and Annaatje Pattiselanno played key roles in the preparation of the material.

References (25)

  • Amaral, D.G. and Witter, M.P. (1995) Hippocampal formation. In G. Paxinos (Ed.), The Rat Nervous System, 2nd edn, Chap....
  • Angelucci, A., Clasca, F. and Sur, M. (1996) Anterograde axonal tracing with the subunit B of cholera toxin: a highly...
  • Brandt, H.M. and Apkarian, A.V. (1992) Biotin-dextran: a sensitive anterograde tracer for neuroanatomic studies in rat...
  • Dolleman-Van der Weel, M.J., Wouterlood, F.G. and Witter, M.P. (1995) Multiple anterograde tracing, combining Phaseolus...
  • Groenewegen, H.J. and Wouterlood, F.G. (1990) Light and electron microscopic tracing of neuronal connections with...
  • Hancock, M.B. (1986) Two-color immunoperoxidase staining: visualization of anatomic relationships between...
  • Lanciego, J.L. and Wouterlood, F.G. (1994) Dual anterograde axonal tracing with Phaseolus vulgaris leucoagglutinin...
  • Lanciego, J.L., Goede, P.H., Witter, M.P. and Wouterlood, F.G. (1996) Use of peroxidase substrate VIP™ for multiple...
  • Mauro, A., Germano, I., Giaccone, G., Giordana, M.T. and Schiffer, D. (1985) 1-Naphthol basic dye (1-NBD), an...
  • Paxinos, G. and Watson, C. (1986) The Rat Brain in Stereotaxic Coordinates, 2nd edn, Academic Press, Sydney,...
  • Rajakumar, N., Elisevich, K. and Flumerfelt, B.A. (1993) Biotinylated dextran: a versatile anterograde and retrograde...
  • Reiner, A., Veenman, C.L. and Honig, M.G. (1993) Anterograde tracing using biotinylated dextran amine, Neurosci. Prot.,...
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