INTRODUCTION

Immunohistochemistry (IHC) is a widely used and powerful technology that enables visual detection of antigens in cells and tissues. Relying on specific and reproducible antibodies, it remains the gold standard for a broad range of applications ranging from basic research to diagnostics.

OVERCOMING IHC CHALLENGESThe lack of standardized guidelines for determining the specificity and functionality of antibodies can lead to varying results between antibodies from different manufacturers. The need for standardization of antibody-based assays and development of antibody validation guidelines is an increasingly important issue and has been highlighted in various recent publications.

In addition to current validation procedures, Bio-Techne has implemented the five pillars of antibody validation, in accordance with recommendations instigated by the International Working Group for Antibody Validation (IWGAV) (Uhlen et al., 2016). These antibody validation principles were developed to be used in an application-specific manner, and to help ensure antibody specificity and reproducibility.

FIVE PILLARS OF ANTIBODY VALIDATION

Dual ISH-IHC Antibody Validation

APPLICATION NOTE

FIGURE 1. The Five Pillars of Antibody Validation. Find out more about at www.novusbio.com/5-pillars-validation

Genetic Strategy Validation- Expression of

the target protein is compared before and after

knockout or knockdown using CRISPR/CAS9 or siRNA/shRNA. If protein

expression following knockout or knockdown is substantially reduced, then

antibody specificity is ensured. View knockout

validated antibodies.

Orthogonal Validation- The target protein is examined

with an antibody independent strategy and

compared with results from an antibody-dependent strategy. A correlation

between these two strategies indicates

specificity between the antibody and its target protein. Examples of

antibody independent techniques may include in

situ hybridization, quantitative PCR, RNA-seq

or mass spectrometry.

Independent Antibody Validation- The data

generated using several antibodies (ideally targeting

different epitopes) in the same protein is compared (e.g. molecular weight and

cellular localization). Consistent results imply

antibody selectivity to the target protein.

Expression of Tagged Proteins Validation- A

tagged protein is used as a standard for comparison in

Western blotting and/or immunocytochemistry

(ICC). For example, if the distribution of the tagged protein overlaps with the

immunofluorescence signal, then antibody

specificity is confirmed.

Biological Strategies Validation- These strategies

use defined biological or chemical modulation of

protein expression to demonstrate antibody specificity to the target

protein. The data is compared across multiple cell lines including positive

and negative expressing cells, and multiple species,

if applicable.

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ORTHOGONAL ANTIBODY VALIDATION - USING THE RNAscopeTM ISH TECHNOLOGY TO VALIDATE ANTIBODIES FOR IHCAnalysis of both RNA and protein provides invaluable information for understanding the regulation of gene expression. Both in situ hybridization (ISH) and IHC assays share common features that make them unique amongst the myriad of protein and RNA detection methods.

• Morphological context: detection of RNA and protein while preserving the cell and tissue structural organization

• Similar workflows: from sample fixation, pre-treatment, hybridization, and signal amplification to data analysis.

Antibody validation techniques for IHC have been addressed by many authors in terms of proper controls, tissue treatment and experimental design. Due to the similarities in workflows, orthogonal antibody validation using the RNAscope ISH assay was addressed by Johns Hopkins School of Medicine (Sfanos KS, et al. Asian Journal of Urology, 2019) and stated to be “invaluable in many of our studies, including helping to validate IHC”.

The utility of RNAscope ISH as an orthogonal method to validate IHC antibodies in drug discovery was also addressed by GSK at the 3rd International Antibody Validation Conference (Goodman et al., 2018). RNAscope was incorporated as a pivotal technology in a high-throughput assay cascade, whereby protein and RNA localization/expression levels were compared in tissues for target identification and validation.

IHC-ISH COMPLEMENTARY WORKFLOWS

FIGURE 2. The workflows for IHC and the RNAscope ISH assay are highly complementary, following four major steps: target retrieval, detection, visualization and analysis.

FIGURE 3. CD68/SR-D1 in Human Tonsil Using Dual RNAscope® ISH and IHC.

CD68/SR-D1 mRNA (red) and protein (green) was detected in formalin-fixed paraffin-embedded tissue sections of human tonsil probed with ACD RNAScope® Probe (Catalog # 560591) followed by immunohistochemistry using R&D Systems Mouse Anti-Human CD68/SR-D1 Monoclonal Antibody (R&D Systems, Catalog# MAB2040) at 5ug/mL for 1 hour at room temperature followed by incubation with the anti-mouse IgG VisUCyte HRP Polymer Antibody (R&D Systems, Catalog # VC001). Tissue was stained using ACD RNAscope® 2.5 HD Duplex Detection Reagents (Catalog # 322500) and counterstained with hematoxylin (blue). Specific staining was localized to cytoplasm in lymphocytes.

SamplePreparation

Immunohistochemistry (IHC)

RNAscope

Target Retrieval

Sample pre-treatment& Permeabilization

AntibodyProbing

Chromogenicor Fluorescent

StainingImage analysis

Sample pre-treatment & Permeabilization

ProbeHybridization

Chromogenicfluorescent signal

amplicfication

Signal analysis and

quantification

Detection Visualization Analysis

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CASE STUDY - USING THE RNAscope ISH ASSAY TO VALIDATE MYC IHC ANTIBODIES Prior attempts to study MYC distribution in human samples have been confounded by a lack of agreement in IHC staining between antibodies targeting the N-terminus and those targeting the C-terminus of the MYC protein. In a study by the Barts Cancer Institute, the RNAscope ISH assay was used to detect MYC mRNA in clinical samples, and to determine the reliability of two different MYC-targeting antibodies (Baker et al., 2016).

IHC was performed on human FFPE normal colon, hyperplastic polyp and neoplastic colon samples using the N-terminally directed antibody Y69 and the C-terminally directed antibody 9E10. The MYC protein distributions were then compared with the location of MYC mRNA, determined by ISH.

FIGURE 4. MYC expression in invasive carcinoma. Representative H&E staining, RNAscope in situ hybridization (MYC mRNA, pink) and immunohistochemical staining with Y69 (N-terminal MYC), 9E10 (C-terminal MYC) and Ki67 antibodies (brown) in a

region of invasive carcinoma. The lower panels (higher magnification images from the upper panels) highlight the reciprocal relationship between the expression of MYC mRNA and the staining with the MYC antibody Y69 and Ki67 antibody , compared with the discordant staining of the 9E10 antibody.

The authors found that the localization of MYC mRNA correlated well with the protein distribution determined with the N-terminally directed antibody Y69 and was also associated with expression of the proliferation marker Ki67. The protein distribution determined with the C-terminally directed antibody 9E10 was not always associated with MYC mRNA, Y69, or Ki67, and often showed a reciprocal pattern of expression, with staining being strongest in non-proliferating cells. The observed discrepancy between the staining patterns suggests that the significance of 9E10 in IHC staining is unclear and should be interpreted with caution.

Baker A.M. et al., Histopathology (2016). 69(2):222-229.

DUAL ISH-IHC RESOURCESTo facilitate the validation of antibodies, protocols combining the RNAscope ISH assays with IHC on the same tissue section (referred to as “Dual ISH-IHC”) have also been developed. This technique can be performed either chromogenically or fluorescently and is compatible with nearly all of the RNAscope assays, including HiPlex, Multiplex Fluorescent, and BaseScope assays. Over 300 publications have utilized this technique in the fields of neuroscience, oncology, stem cells, and beyond1-24.

For more information on the dual ISH-IHC technique, including tech notes and guidelines, please visit: https://acdbio.com/science/applications/research-solutions/dual-ish-and-ihc

For a full list of Dual ISH-IHC publications, please visit: www.acdbio.com/science/scientific-resources/publications/publication

For labs that require high throughput and automated analysis, ACD has partnered with Leica Biosystems and Roche Tissue Diagnostics, Inc to deliver RNAscope ISH assays on these automated stainers, both of which offer the possibility to combine ISH and IHC on the same slide.

H&E MYCmRNA Y69 9E10 Ki67

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DUAL ISH-IHC VALIDATED ANTIBODIES FROM BIO-TECHNEAs the home of the RNAscope in situ hybridization technology and a range of >34,000 antibodies for IHC, Bio-Techne is uniquely able to support researchers using RNA ISH and IHC techniques. The following antibodies from R&D Systems and Novus Biologicals have been validated for dual ISH-IHC assays by our ISH-IHC experts.

Table of Selected Dual ISH-IHC Validated Antibodies

Marker Catalog # (Clone) RNAscope ISHCompatibility

CD8 NB100-65729 (4B11) 3

CD45 NBP2-29631 (UCHL-1) 3

CD52 NBP2-52645 (Campath-1H) 3

CD68 MAB2040 (298813) 3

CD163 NBP2-36494 (6E10.1G6) 3

FoxP3 MAB8214 (1054C) 3

GATA-3 MAB6330 (634913) 3

Vimentin MAB21052 (979517) 3

Cytokeratin 19 MAB35061 (963420) 3

α-Smooth Muscle Actin MAB1420 (1A4) 3

Caspase-3 MAB7071 (1112I) 3

CD14 NBP2-37291 (4B4F12) 3

CD31 NB600-562 (JC/70A) 3

Von Willebrand Factor NB600-586 (polyclonal) 3

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AN_ISH-IHC_RSDFY20-8396

1. Zahr, 10.1016/j.neuron.2017.12.045

2. Periyasamy, 10.1523/jneurosci.3474-17.2018

3. Fielding, 10.1113/jp275996

4. Mao, 10.1186/s13058-018-1057-0

5. Cassetta, 10.1016/j.ccell.2019.02.009

6. Norman, 10.1371/journal.pone.0216795

7. Albe, 10.1371/journal.ppat.1007833

8. Sampson, 10.1002/ijc.31316

9. Rodda, 10.1016/j.immuni.2018.04.006

10. De Schepper, 10.1016/j.cell.2018.07.048

11. Duan, 10.1016/j.neuron.2018.08.030

12. Chan, 10.1371/journal.pone.0207619

13. Jakel, 10.1038/s41586-019-0903-2

14. Zhang, 10.1016/j.celrep.2019.01.056

15. Bartesaghi, 10.1016/j.celrep.2019.02.098

16. Karpus, 10.1016/j.celrep.2019.02.101

17. Gerhart, 10.1371/journal.pone.0214758

18. Sorrells, 10.1038/s41467-019-10765-1

19. Bertram, 10.1038/s41467-019-10697-w

20. Nikolakopoulou, 10.1038/s41593-019-0434-z

21. Smillie, 10.1016/j.cell.2019.06.029

22. Tokue, 10.1016/j.stemcr.2017.01.006

23. Majumdar, 10.1007/s00125-019-4967-1

24. McGary, 10.1016/j.immuni.2017.09.018

SELECTED REFERENCES USING BIO-TECHNE ANTIBODIES (DOI #S ARE LISTED)