Improved immunocytochemical identification of neural, endothelial, and inflammatory cell types in paraffin-embedded injured adult rat spinal cord

https://doi.org/10.1016/j.jneumeth.2004.04.008Get rights and content

Abstract

Methods that facilitate the accurate counting of specific neural cell types would be of substantial value in evaluating the efficacy of treatments applied to spinal cord injury. This report describes reliable procedures for identification of neurons, oligodendrocytes, astrocytes, endothelial cells and inflammatory cells (neutrophils and activated macrophage/microglial cells) in paraformaldehyde-fixed, paraffin-embedded injured adult rat spinal cord. Antigen retrieval techniques (enzymatic and thermal) were used to improve antibody access to masked epitopes. To decrease background immunofluorescence and autofluorescence of hemoglobin, the tissue sections were pretreated with 0.1% sodium borohydride in PBS (30 min), followed by 1–5 min incubation in 0.5% Sudan black in 70% ethanol. Commercially available techniques to amplify the primary signal such as tyramide signal amplification (TSA) and avidin/biotin/peroxidase/DAB/nickel/cobalt amplification (ABP/DABA) were also tested. Hoechst 33342 nuclear staining was used to indicate cell location, number, and integrity, thereby avoiding misidentification of cells. The best antibodies were: anti-NeuN antibody for neurons, anti-S100 for astrocytes, and anti-S100 and APC-7 antibodies in combination for oligodendrocytes, anti-laminin (LN) for endothelial cells, and ED1 antibody for activated macrophages and microglia. Amplification of the primary signal with TSA or ABP/DABA was also found to be beneficial.

Introduction

The quantitative assessment of neural injury and the efficacy of strategies for neural protection and repair remain problematic in spinal cord injury research. Quantitation of specific neural cell types in the injury area and away from the injury is an effective measure of tissue sparing and is important for assessing the impact of the injury and treatments for different cell populations. For this task the availability of reliable histological techniques to identify cells and cellular structures is critical.

Due to the advantages of simple storage, long-term preservation and easy sectioning, paraffin embedding is preferred by many to other tissue preparations. Compared with frozen tissue, however, a much more limited number of commercially available antibodies has been proven to work on paraffin-embedded sections. Furthermore, many that do work need to be used at higher concentrations, increasing background staining, cost, and the possibility of error in quantitation. Another problem is background immunofluorescence, which has many sources, such as autofluorescence of pigments and tissue fixed in aldehydes. The use of fluorescent probes in the analysis of tissue immediately after injury is greatly limited by the presence of hemoglobin (Aubin, 1979, Benson et al., 1979); hemoglobin autofluorescence may mask or be mistaken for the primary signal. To overcome autofluorescence of lipofuscin-like products in fluorescently labeled nervous tissue, Schnell et al. (1999) pretreated tissue sections with 1–10 mM CuSO4 in 50 mM ammonium acetate buffer or with 1% Sudan black in 70% ethanol with good results. Tissue autofluorescence resulting from fixation with aldehydes, such as paraformaldehyde, and especially glutaraldehyde, can be significantly decreased by a 30 min incubation in 0.1% sodium borohydride in PBS (Bacallao et al., 1995). In addition to producing some background immunofluorescence, formalin fixation may mask the epitopes of some antigens, significantly decreasing or even abolishing specific staining. Various antigen retrieval techniques are available to unmask these epitopes altered by paraformaldehyde treatment (Evers et al., 1998, McNicol and Richmond, 1998, Shi et al., 1991, Shi et al., 1998).

This paper describes a series of optimized histological procedures proven to increase the primary antibody signal [high performance primary antibodies, antigen retrieval techniques, amplification techniques using tyramide signal amplification (TSA) and avidin/biotin/peroxidase/DAB/nickel/cobalt amplification (ABP/DABA) kits] and treatment to significantly decrease autofluorescence (0.1% sodium borohydride and 0.5% Sudan black incubation) and background staining (blocking and wash solutions). Although 19 antibodies were tested, only five were found to allow specific identification of the major cell types present in injured spinal cord. Finally, the use of nuclear staining (Hoechst 33342) in combination with cell-specific antibodies allowed the precise identification of intact cells as opposed to cell ghosts, thereby improving the reliability of the counting method. The use of these protocols and antibodies provided consistent and reproducible staining; these methods have been used for identification and counting of neurons, oligodendrocytes, astrocytes, endothelial cells, and activated macrophage/microglial cells in a study to analyze paraformaldehyde-fixed, paraffin-embedded adult rat spinal cord following injury. The results of this study will be reported separately.

Section snippets

Materials and methods

Eight adult female Fischer rats (200 g), included in a study of angiogenesis following moderate contusion injury of the spinal cord (Casella et al., 2002), provided tissue for sections on which the procedures were tested. For histological examination, a three cm segment of spinal cord containing the injury site was removed following perfusion with 4% buffered paraformaldehyde (Casella et al., 2002). Spinal cords were further fixed overnight in the same fixative, immersed one day in PBS and then

Selection of effective antibodies to immunostain paraffin-embedded tissue

By careful application of the techniques described in Section 2, several antibodies were found to be effective in staining paraffin sections; in fact, each of the major cell types in injured spinal cord could be clearly identified and quantitated in these sections. Antibodies that did not stain after antigen retrieval and ABP/DABA techniques were considered inadequate. The results of our survey are summarized in Table 1.

Large- and medium-sized neurons in the ventral horn were easily visualized

Discussion

The major contributions of this study are: (1) the selection of a combination of antibodies that allow identification of neurons, oligodendrocytes, astrocytes, endothelial cells, activated macrophages/microglia and neutrophils in paraformaldehyde-fixed, paraffin-embedded injured adult rat spinal cord; (2) the combination of nuclear staining with primary antibody staining as a means of facilitating the identification and counting of cells; (3) the use of techniques (i.e. the Sudan black

Acknowledgements

We thank Susan Kraydieh for help with histology, Beata Frydel for imaging advice, Rob Camarena for figure formatting, and Diana Masella for word processing. This work was supported by The Miami Project to Cure Paralysis and NIH NS 38665. GTBC was a Lois Pope LIFE Fellow.

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