Application of Immunohistochemistry in Stereology for Quantitative Assessment of Neural Cell Populations Illustrated in the Göttingen Minipig

Abstract

BackgroundStereology is the study of estimating geometric quantities. When successfully applied, the combination of immunohistochemistry (IHC) and stereology eliminates intra- and interobserver variability for cell type identification.Methodology/Principal FindingsWe propose a method to validate existing antibody based cell type markers for stereological application. Comparison was made on the 100-days-old Göttingen minipig (G-mini) neocortex between estimates of total neuron number derived from Giemsa staining using morphological criteria and immunohistochemistry-based cell counting with NeuN. The mean total neuron numbers estimated by the two staining methods were not significantly different. Estimated quantities, including glial cell number, neocortical volume, cell densities and glial-to-neuron ratio were also presented. Additionally, we assessed other commonly used glial markers and discussed how to evaluate the advantages and disadvantages of these markers for stereological estimation of cell number.Conclusion/SignificanceThe concordance in quantitative estimates of total neuron number derived from NeuN- and Giemsa-stained sections provides evidence for the sensitivity and specificity of NeuN as a neuronal marker in the G-mini. Although time-consuming, quantitative validation of IHC should always be considered in stereological studies if there is doubt of the sensitivity, specificity, or reproducibility of cell type markers. Inaccurate staining may cause both over- and underestimation of the total cell number and inflict considerable limitation when analyzing the results.

Distinction of Neurons, Glia and Endothelial Cells in the Cerebral Cortex: An Algorithm Based on Cytological Features

“…Others have estimated neuron and glial cell number without distinction of glial types (Ongur et al, 1998; Rajkowska et al, 1999; Selemon et al, 1999; Cotter et al, 2001; Dombrowski et al, 2001; Lidow and Song, 2001; Christensen et al, 2007). Only one of these studies describes briefly the features of neuron and glial cell types for Nissl stain and confirms their cytological findings with immunohistochemistry (Hou et al, 2012). Most articles contain only brief descriptions and base the distinction of neurons from glial cells on the presence of a distinct nucleolus, which can be misleading because small neurons frequently have small nucleoli surrounded by thick perinucleolar heterochromatin clumps and some large neurons may have two nucleoli.…”

“…Only two modern studies describe in detail cell cytology in the brain of rats using semithin sections stained for toluidine blue (Ling et al, 1973; Gabbott and Stewart, 1987). Another study described briefly neuron and glial cell features in the human cerebral cortex stained for Nissl (Pelvig et al, 2008) and in another article, the same group confirmed their cytological findings with immunohistochemistry (Hou et al, 2012). Thus, there is a lack of detailed, updated, systematic and well-illustrated descriptions of the cytology of neurons and glial cell types, especially in the primate brain.…”

A stereological study of the mediodorsal thalamic nucleus in Down syndrome

“…The second population of neurons consists of larger cells that are mostly excitatory relay neurons projecting to the cerebral cortex (Armstrong, 1990;Dorph-Petersen et al, 2004). Clear identification of the two types of neurons is only possible using immunocytochemical techniques, which were not used in this study as the accuracy of Nissl staining-based estimates are comparable to the antibody-based estimates (Lyck et al 2006;Hou et al 2012). Thus, differentiation between large (projection) neurons and small (inhibitory) neurons was based only on morphology and size (Fig.…”

Antibody Incubation at 37°C Improves Fluorescent Immunolabeling in Free-Floating Thick Tissue Sections

“…In brief, after extensive washing with PBS-G (0.3 M glycine in 0.01 M PBS, pH 7.4) at RT, free-floating thick tissue sections were preconditioned for tissue loosening: ( i ) skin: neat DMSO for 15 min to loosen collagen structures in the upper dermis (UD) and dense cellular networks in the epidermis (10); ( ii ) spinal cord: antigen retrieval with sodium citrate solution (0.01 M sodium citrate, 0.05% Tween-20, pH 6.0) at 80C for 45 min to improve antigenic reactivity and antibody penetration (9,11); and ( iii ) sciatic nerve: thermally-assisted delipidation with 0.2 M sodium dodecyl sulfate (SDS) (in PBS, pH 9.0) at 37C for 3 h (7). After extensive washing with PBST (0.2% Tween-20 in 0.01 M PBS, pH 7.4) at RT, thick tissue sections were then permeabilized with PBS-Tx (0.3% Triton X-100 in 0.01 M PBS, pH 7.4): 4 15 min for skin and spinal cords, and 6 15 min for sciatic nerves.…”

“…However, due to poor antibody penetration, standard protocols for fluorescent immunolabeling of free-floating thick tissue sections had an unsatisfactory labeling quality (low sensitivity for sparsely or moderately expressed proteins; faint or negative immunolabeling in the center of thick tissue sections and high non-specific binding) and an inadequate labeling efficiency (requiring high antibody concentrations and long antibody incubation times). These drawbacks render immunolabeling far from ideal for 3-D in situ quantitative biology applications (2,8,9,13). …”