Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Summary

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

Abstract

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Introduction

Immunohistochemistry (IHC) is a standard lab technique for detection of protein within tissue, and IHC is still widely used in both research and diagnostic pathology. The evaluation of IHC staining is often semi-quantitative, introducing potential bias into interpretation of results. Many semi-quantitative approaches have been developed which incorporate both staining intensity and staining extent into final diagnosis ^1-4. Other systems include scoring intensity and subcellular location in order to better localize expression ⁵. Incorporation of average scores from multiple viewers is often utilized in order to minimize the effects of single viewe....

Protocol

     NOTE: The protocol herein describes staining and analysis of a tissue microarray (TMA), described previously ^12,17,18. The 4 µm thick TMA section was obtained from a paraffin block using a standard microtome. 

     NOTE: A spectral library for the 4 chromogens and counterstain should be created for image quantitation. In order to do this, the optimized protocol for each individual antibody should be run with one antibody per slide, minus the final counterstain. A fifth slide should be stained with hematoxylin to generate the 5 images needed to create the spectral library.

1. Multiplex Immunohisto....

Results

In Figure 1, training is performed on prostate tissues to segment images into epithelial and stromal portions, along with the non-tissue compartment. By using the epithelial membrane marker E-cadherin, cell segmentation was performed to separate the nucleus, cytoplasm, and membrane portions, shown in Figure 2.

In one experiment, we used multiplexed IHC to investigate the expression and localizat.......

Discussion

The use of traditional immunohistochemistry for evaluating protein expression is limited by subjective, semi-quantitative methods of analysis ^22,23. Advance platforms have been created for high-throughput analysis of biomarker expression and localization. Detailed segmentation of both tissue and subcellular compartments allows users to study biomarker expression, localization, and co-localization with other markers of interest. In previous studies, we have demonstrated the utility of IHC and multispectral imag.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Xylene	Fisher Chemical	X3F1GAL	NFPA rating: Health – 2, Fire – 3 , Reactivity – 0
Ethyl Alcohol-200 proof	Fisher Scientific	4355223	NFPA rating: Health – 0, Fire – 3 , Reactivity – 0
Tris Base	Fisher Scientific	BP152-500	NFPA rating: Health – 2, Fire – 0 , Reactivity – 0
Tris Hydroxymethyl aminomethane HCl	Fisher Scientific	BP153-1	NFPA rating: Health – 2, Fire – 0 , Reactivity – 0
Tween 20	Chem-Impex	1512	NFPA rating: Health – 0, Fire – 1 , Reactivity – 0
Phosphate-buffered saline	Fisher Scientific	BP2944-100	NFPA rating: Health – 1, Fire – 0 , Reactivity – 0
Peroxidazed	Biocare Medical	PX968	Avoid contact with skin and eyes. May cause skin irritation and eye damage.
Estrogen Receptor alpha	Thermo Fisher Scientific-Labvision	RM9101	Not classified as hazardous
Androgen Receptor	SCBT	sc-816	Not classified as hazardous
CD147	Biodesign	P87535M	Not classified as hazardous
E-cadherin	Dako	M3612	Not classified as hazardous
Renoir Red Andibody Diluent	Biocare Medical	PD904	It is specially designed to work with Tris-based antibodies
DeCloaking Chamber	Biocare Medical	Model DC2002	Take normal precautions for using a pressure cooker
Barrier pen-Immuno Edge	Vector Labs	H-4000
Denaturing Kit-Elution step	Biocare Medical	DNS001H	Not classified as hazardous
Mach 2 Goat anti-Rabbit HRP Polymer	Biocare Medical	RHRP520	Not classified as hazardous
Mach 2 Goat anti-Rabbit AP Polymer	Biocare Medical	RALP525	Not classified as hazardous
Mach 2 Goat anti-Mouse HRP Polymer	Biocare Medical	M3M530	Not classified as hazardous
Betazoid DAB Chromogen Kit	Biocare Medical	BDB2004	1. DAB is known to be a suspected carcinogen. 2. Do not expose DAB components to strong light or direct sunlight. 3. Wear appropriate personal protective equipment and clothing. 4. DAB may cause sensitization of skin. Avoid contact with skin and eyes. 5. Observe all federal, state and local environmental regarding disposal
Warp Red Chromogen Kit	Biocare Medical	WR806	Corrosive. Acid that may cause skin irritation or eye damage.
Vina Green Chromogen Kit	Biocare Medical	BRR807	Harmful if swallowed
Bajoran Purple Chromogen Kit	Biocare Medical	BJP807	Flammable liquid. Keep away from heat, flames and sparks. Harmful by ingestion or absorption. Avoid contact with skin or eyes, and avoid inhalation.
Cat Hematoxylin	Biocare Medical	CATHE	Purple  solution  with  a  mild  acetic  acid  (vinegar)  scent.  May  be  irritating  to  skin  or  eyes.  Avoid  contact  with  skin  and  eyes.  Avoid  ingestion.
XYL Mounting Media	Richard Allen	8312-4	NFPA rating: Health – 2, Fire – 3 , Reactivity – 0
1.5 Coverslips	Fisher Brand	22266858	Sharp edges
Incubation (Humidity)Chamber	obsolete	obsolete	Multiple vendors available
Convection Oven	Stabil- Therm	C-4008-Q
Background Punisher Blocking Reagent	Biocare Medical	BP974	This product is not classified as hazardous.
inForm software	PerkinElmer	CLS135781	Primary multispectral imaging software used in manuscript
Nuance software	PerkinElmer	Nuance EX	Software used for making spectral libraries within manuscript
Vectra microscope and slide scanner	PerkinElmer	VECTRA	Automated slide scanner and microscope for obtaining IM3 image cubes