Abstract
We describe a novel approach for quantification and colocalization of immunofluorescence (IF) signals of multiple markers on high-resolution panoramic images of serial histological sections utilizing standard staining techniques and readily available software for image processing and analysis. Human gingiva samples stained with primary antibodies against the common leukocyte antigen CD45 and factors related to heparan sulfate glycosaminoglycans (HS GAG) were used. Expression domains and spatial gradients of IF signals were quantified by histograms and 2D plot profiles, respectively. The importance of histomorphometric profiling of tissue samples and IF signal thresholding is elaborated. This approach to quantification of IF staining utilizes pixel (px) counts and comparison of px grey value (GV) or luminance. No cell counting is applied either to determine the cellular content of a given histological section nor the number of cells positive to the primary antibody of interest. There is no selection of multiple Regions-Of-Interest (ROIs) since the entire histological section is quantified. Although the standard IF staining protocol is applied, the data output enables colocalization of multiple markers (up to 30) from a given histological sample. This can serve as an alternative for colocalization of IF staining of multiple primary antibodies based on repeating cycles of staining of the same histological section since those techniques require non standard staining protocols and sophisticated equipment that can be out of reach for small laboratories in academic settings. Combined with the data from ontological bases, this approach to quantification of IF enables creation of in silico virtual disease models.
Highlights
The immunofluorescence (IF) (and immunohistochemistry (IHC) in general) has long been recognized as one of the fundamental methods for biomedical research
To demonstrate the mechanics of the approach, we used human gingiva samples stained with primary antibodies against cell surface heparan sulfate proteoglycan (HSPG) syndecan 1 (Sdc1), heparan sulfate glycosaminoglycan (HS GAG), HS GAG-biosynthesis proteins and common leukocyte antigen CD45
The substantial difference of group mean values of fraction areas of tissue compartments presents a significant bias for statistical analysis of expression domains of IF signals between groups of samples if they, for instance, belong to a marker whose expression is more restricted to a particular tissue compartment—a good example is Sdc[1], which is mainly expressed by epithelial cells and usually referred to as the epithelial HSPG (Heparan Sulfate ProteoGlycan)[7]
Summary
The immunofluorescence (IF) (and immunohistochemistry (IHC) in general) has long been recognized as one of the fundamental methods for biomedical research. There are software tools for quantification of all three of these properties, but while the conventional computer-assisted scoring systems for quantification of staining are able to turn the expression patterns and expression domains into quantifiable parameters, they still fall short on quantification of IF signals’ spatial gradients[3] This does not pose much of a problem for quantification of protein-markers expressed in the well-defined cell compartments (cell nuclei), or when their expression is localized to specific tissue structures and able to be analyzed within smaller Regions-Of-Interest (ROIs) determined by investigator[4]. The quantification of IF signals from ubiquitously expressed markers with non-nuclear expression patterns requires different approach This is due to their generally large expression domains (which in conventional approach would necessitate the selection of increasing number of ROIs), and because their spatial gradients are extremely important indicator of biological function[5,6]. This can serve as an alternative for colocalization of IF staining of multiple markers based on repeating cycles of staining of the same histological section since those techniques require non standard staining protocols and sophisticated equipment that can be out of reach for small laboratories in academic settings
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