Synergistic Tissue Counterstaining and Image Segmentation Techniques for Accurate, Quantitative Immunohistochemistry

Abstract

Quantitative analysis of digitized IHC-stained tissue sections is increasingly used in research studies and clinical practice. Accurate quantification of IHC staining, however, is often complicated by conventional tissue counterstains caused by the color convolution of the IHC chromogen and the counterstain. To overcome this issue, we implemented a new counterstain, Acid Blue 129, which provides homogeneous tissue background staining. Furthermore, we combined this counterstaining technique with a simple, robust, fully automated image segmentation algorithm, which takes advantage of the high degree of color separation between the 3-amino-9-ethyl-carbazole (AEC) chromogen and the Acid Blue 129 counterstain. Rigorous validation of the automated technique against manual segmentation data, using Ki-67 IHC sections from rat C6 glioma and beta-amyloid IHC sections from transgenic mice with amyloid precursor protein (APP) mutations, has shown the automated method to produce highly accurate results compared with ground truth estimates based on the manually segmented images. The synergistic combination of the novel tissue counterstaining and image segmentation techniques described in this study will allow for accurate, reproducible, and efficient quantitative IHC studies for a wide range of antibodies and tissues.

Neuropathologic Heterogeneity Does Not Impair Florbetapir-Positron Emission Tomography Postmortem Correlates

“…These blocks were embedded in paraffin and immunohistochemically stained for β-amyloid using the anti-Aβ antibody 4G8 (Covance, Emeryville, CA)(8). The cortical amyloid burden on each of these slides, which included all morphologically-defined plaque subtypes, was defined as the percentage of gray matter occupied by stained neuropil exceeding a threshold stain density (8, 21), using PERMITS™ image processing and analysis software (Biospective Inc., Montreal, Quebec, Canada). Amyloid burden estimates were thus obtained for all six cortical regions of interest (ROIs), and a mean cortical amyloid load was determined by averaging these.…”

Correlation of Amyloid PET Ligand Florbetapir F 18 Binding With Aβ Aggregation and Neuritic Plaque Deposition in Postmortem Brain Tissue

“…The sections were counterstained with Acid Blue 129 and coverslipped using an aqueous mounting media. 23 The stained slides were digitized using a Zeiss MIRAX high-resolution, automated slide scanner (Carl Zeiss Canada, Toronto, ON) and the digital images were converted from MIRAX to MINC (Medical NetCDF) file format. Image quantification was performed using the PERMITS ™ image processing/analysis software (Biospective Inc., Montreal, Canada).…”

Quantitative Magnetic Resonance Imaging of Cortical Multiple Sclerosis Pathology

“…IHC slides were counterstained for 1 minute in 0.1% Acid Blue 129 (Sigma-Aldrich Canada; Oakville, ON, Canada), rinsed for 30 s in sodium acetate-acetic acid buffer (pH 3.6), and air-dried. The slides were mounted in a specially-formulated aqueous mounting media [41]. …”

“…The myelin fibers in the MBP-stained sections were segmented from the background counterstain using an automated image segmentation algorithm based on the high degree of color separation between the red-brown AEC chromogen and the light blue counterstain [41]. The resulting binary maps of myelin fibers were down-sampled in-plane by averaging the pixels to match the resolution of the qMR images, thereby generating a 2D map of myelin content as a percentage of local area.…”