Application Note Drug Safety Assessment

Robust detection of RNA biomarkers for drug safety assessment in preclinical animal models by fully automated RNAscope® 2.5 LS Assay

Ming-Xiao He1, Bingqing Zhang1, Daniel Kim1, Tania Franks2, Marc Roy2, Chris Bunker1, Yuling Luo1, Xiao-Jun Ma1, Emily Park1

Preclinical drug safety Abstract eosin (H&E) stain, and also for evaluation of assessment in animal specific biomarkers by immunohistochemistry models is used to evaluate Robust assays to evaluate biomarkers in tissue (IHC). IHC assay has been commonly applied to the pathological effects are needed for preclinical safety assessment and assess therapeutic targets and toxicity-related induced by novel therapeutic toxicity studies. Here we present the application biomarkers. However, consistent and systematic ® agents. Here we present the of the fully automated RNAscope 2.5 LS Assay application of IHC techniques has been hindered use of the RNAscope® 2.5 on Leica BOND RX for RNA in situ hybridization in by inconsistent performance of various antibody LS Assay for the evaluation formalin-fixed paraffin-embedded (FFPE) tissues clones, time-consuming antibody development of biomarkers in tissues from three commonly used animal models (rat, and validation, and general lack of reagents for from three preclinical animal cynomolgus monkey, and dog). We demonstrate some animal models. RNA in situ hybridization models. In this study we: robust assay performance with high signal-to- (ISH) technology presents an attractive alternative noise ratio and well-maintained morphology in 25 method for pathological evaluation of biomarkers • Identify optimal pretreatment different tissues from each species. Based on conditions for different tissues in tissues from various preclinical animal models, these tests, we provide recommendations for in different species because nucleic acid-based probes specific for proper control for sample qualification of any biomarkers associated with drug toxicity • Provide recommendations each tissue type, as well as optimal pretreatment or mechanism can be developed and for control gene selection for protocol selection. For specific target RNA validated rapidly. tissue qualification markers, we successfully detected cell type ® specific markers such as CD31 (PECAM1) and The RNAscope technology, an advanced • Detect specific RNA markers CD68, proliferation marker Ki-67 (MKI67), and cell platform for in situ RNA detection, enables in various FFPE tissues from cycle marker Cyclin E1 (CCNE1), as well as detection of almost any RNA biomarker with multiple species -related molecules Puma (BBC3), Fas single-molecule detection sensitivity and high (CD95), and DR5 (TNFRSF10B). This study specificity in formalin-fixed paraffin-embedded 2 demonstrates that the RNAscope® 2.5 LS Assay (FFPE) tissues . It provides a universal solution can be an attractive platform for biomarker to characterize tissue distribution of drug targets analysis in tissues for preclinical safety and biomarkers in a highly specific and sensitive assessment and general animal studies. manner, without the need to wait for antibody development and validation. The RNAscope® assay can be performed in fully automated Introduction staining systems, including Ventana Discovery Preclinical drug safety assessment in animal XT, Ventana Discovery ULTRA, and Leica BOND models has been well established as a routine RX instruments. The assay allows visualization of laboratory practice to evaluate the pathological each individual RNA molecule as a punctate dot alterations induced by novel therapeutic agents1. under a standard bright field microscope. The RNA This preliminary evaluation serves a major role dots can be quantified by counting the number of in the development of new treatments prior to signal dots in individual cells, either manually or trials in humans. Histopathological techniques by image analysis tools, including HALOTM (Indica have been traditionally applied for general Labs) and SpotStudioTM (ACD) software. morphological evaluation by hematoxylin and In this study, we demonstrate the feasibility

1 Advanced Cell Diagnostics, Inc 3960 Point Eden Way Hayward, CA, USA 94545 2Drug Safety Research and Development, Pfizer Global Research and Development, Groton, CT 06340, USA 1 of evaluating RNA biomarkers in 25 types of tissues from three Automated RNAscope® 2.5 LS assay commonly used animal models using the RNAscope® 2.5 LS Reagent ® Kit-BROWN on the Leica BOND RX instrument. Robust RNA detection Ready-to-use reagents from RNAscope 2.5 LS Reagent Kit-BROWN was achieved following a standard protocol in almost all of the were loaded onto the Leica BOND RX instrument according to the tissues tested, with minor alterations to the prestaining conditions user manual (Doc. No. 322100-USM). FFPE tissue sections were for a few tissues. Here, we identified the threshold of pretreatment baked and deparaffinized on the instrument, followed by needed for different tissue types. We also provide recommendations retrieval (using Leica Epitope Retrieval Buffer 2 at 95°C or at 88°C for for control probes to be applied for tissue qualification, and present 15 min) and protease treatment (15 min at 40°C). Probe hybridization, the evaluation of RNA biomarkers including cell type specific markers signal amplification, colorimetric detection, and counterstaining ® (CD68 and endothelial marker PECAM1), proliferation marker Ki-67 were subsequently performed. A schematic of the RNAscope 2.5 LS (MKI67), and cell cycle marker Cyclin E1 (CCNE1). Overall, our study Assay workflow on Leica BOND RX is presented in Figure 1A. shows that the fully automated RNAscope® 2.5 LS Assay is capable of detecting a broad range of RNA targets in all major tissue types RNAscope® probes with little to no optimization needed, and thus well suited for the Control probes of low-, medium-, and high-expressing housekeeping histopathological evaluation of biomarkers in the assessment of (POLR2A, PPIB, and UBC, respectively) were designed and drug-derived toxicity in various tissues and animal models. tested for tissues from each species (Table 2). Because the sequences of the human probes for housekeeping genes are over Materials and Methods 95% homologous to the respective target mRNA sequences of FFPE tissues cynomolgus monkey, human probes were used to test samples of cynomolgus monkey. The bacterial probe DapB was used as a Multiple tissues from three commonly used animals (rat, dog, and negative control. Probes for the cell type biomarkers, proliferation cynomolgus monkey) were harvested using a standard protocol markers, and apoptosis-related molecules used in this study were at the drug safety research and development laboratory of Pfizer- designed for each species. As summarized in Table 3, species- Groton (Table 1). Tissues were cut into 3 mm thickness then fixed specific target probes were tested for all RNA targets except two in 10% neutral-buffered formalin (NBF) for 24-48 hours. Fixed genes, CD68 and KI67, for which human probes were used to detect tissues were dehydrated in a graded series of ethanol and xylene, cynomolgus monkey genes, due to 90-95% homology between the followed by infiltration of melted paraffin at 56°C in an automated probe sequence and target mRNA sequence. processor. Tissue microarrays (TMAs) were constructed, sectioned at a thickness of 5 µm and mounted on the SuperFrost® Plus slides Image acquisition and data analysis (Fisherbrand Cat # 12-550-15). Images were acquired using a Leica Biosystems Aperio AT2 Digital Pathology Scanner. RNA markers were analyzed based on

Animal Models: Rat, Dog, and Cynomolgus the average RNA dot number per cell. RNA quantity was scored based on manual counting described as follows. Staining results Hematopoietic Thymus, Lymph Node, Spleen, Tonsil were categorized into five grades according to the number of dots system visualized under a bright-field microscope. 0: No staining or less than GI tract Esophagus, Stomach, Duodenum, Jejunum, Colon 1 dot to every 10 cells (40X magnification); 1+: 1-3 dots/cell (visible at 20-40X magnification); 2+: 4-10 dots/cell, very few dot clusters Urinary tract Kidney, Urinary bladder (visible at 20-40X magnification); 3+: >10 dots/cell, and less than 10% Reproductive system Epididymis, Prostate, Testis, Ovary positive cells have dot clusters (visible at 20X magnification); and 4+: >10 dots/cell, and more than 10% positive cells have dot clusters Skin/soft tissues Skin, Skeletal muscle (visible at 20X magnification).

Endocrine glands/ Liver, Pancreas, Adrenal gland exocrine glands Results Respiratory system Lung, Bronchus Optimal pretreatment condition for Nervous system Spinal cord, Retina different tissues in different species

® Cardiovascular The standard protocol of RNAscope 2.5 LS Reagent Kit is designed Heart system to work for the majority of FFPE tissues. In this study, to achieve optimal detection of RNA molecules in each tissue type, we compared

TABLE 1. Tissue types from three commonly used animal models. two different pretreatment conditions, standard and mild, with a modification in the epitope retrieval step (Figure 1A). The standard

2 Application Note B Cynomolgus monkey - Testis A RNAscope 2.5 LS Assay workflow Standard Mild √ Steps Description

Deparaffinization

Epitope retrieval (ER2 95º C/88º C) Hs-POLR2A

Pretreat Protease (40º C)

H2 O2 block

Target Probe Hybridization

Hybridize DapB AMP1

AMP2 Rat - Liver AMP3 Amplify Standard √ Mild

AMP4

AMP5

AMP6 Rn-Ppib

DAB reaction

Hematoxylin stain Stain and detect

Image detection under standard DapB microsope/scanner

C D Default Recommendation Optimal Protocol: Dog, and Optimal Protocol: Rat Cynomolgus monkey

Hard to access Hard to access

Heart, liver, esophagus, skin Heart, liver, esophagus, skin

ER2 Spinal cord, stomach, kidney, pancreas, ER2 Spinal cord, stomach, kidney, pancreas, 95º C lung, urinary bladder, skeletal muscle 95º C lung, urinary bladder, skeletal muscle

Duodenum, jejunum, colon, ovary, prostrate, Duodenum, jejunum, colon, ovary, prostrate, epididymis, testis, adrenal gland epididymis, testis, adrenal gland

ER2 Retina, spleen, lymph node, tonsil, thymus ER2 Retina, spleen, lymph node, tonsil, thymus 88º C 88º C

FIGURE 1. Selection of optimal pretreatment for RNAscope® 2.5 LS Assay. (A) RNAscope® 2.5 LS Assay workflow on Leica BOND RX instrument. (B) Examples of the optimal pretreatment protocol. ISH results from cynomolgus monkey testis and rat liver FFPE tissues that were processed by two different pretreatment protocols (standard and mild) and probed with either Hs-POLR2A, Rn-Ppib, or DapB. (C) Default pretreatment recommendation for different tissue types and the optimal pretreatment conditions for tissues from rat examined in this study. (D) Optimal pretreatment conditions for tissues from dog and cynomolgus monkey examined in this study. Tissues sharing the same pretreatment protocol may be grouped together for tissue microarray. Note: Optimal pretreatment is based on FFPE tissue prepared by Pfizer-Groton. Optimization may be required for samples prepared and processed differently.

Drug Safety Assessment 3 pretreatment condition used BOND Epitope Retrieval Buffer 2 (ER2) Species Probe Name Alias of Targets Catalog No. at 95°C for 15 min, followed by protease digestion at 40°C for 15 min. Name The mild epitope retrieval condition used ER2 at 88°C for 15 min and Cynomolgus 2.5 LS Probe – Mfa-BBC3 JFY1; PUMA; JFY-1 434458 the same protease digestion step. monkey The optimal pretreatment condition for each tissue type was Cynomolgus 2.5 LS Probe – Mfa-CCNE1 cyclin E1 434468 determined based on RNA signal level and the integrity of monkey morphology. Examples of tissues showing different staining results Homo GP110, LAMP4, between standard and mild conditions are shown in Figure 1B. 2.5 LS Probe – Hs-CD68 560598 sapiens To increase the success rate of the initial RNAscope® ISH test, it SCARD1 is generally recommended to start with the default pretreatment ALPS1A, APO-1, setting, in which lymphoid tissues and retina are treated with BOND Cynomolgus 2.5 LS Probe – Mfa-FAS APT1, CD95, 434488 ER2 at 88°C and other tissues are treated with BOND ER2 at 95°C monkey FASTM, TNFRSF6 (Figure 1C). This setting worked best for all of the rat tissues tested. However, the mild pretreatment was the optimal condition for several Homo Ki-67, KIA, MIB-, 2.5 LS Probe – Hs-MKI67 591778 other tissues from dog and cynomolgus monkey, as shown in Figure Sapiens MIB-1, PPP1R105 1D. Complete data with tissue images and recommended conditions for all samples are available in the Appendix. The background signal, CD31, EndoCAM, Cynomolgus 2.5 LS Probe – Mfa-PECAM1 GPIIA’, PECA1, 434498 evaluated by the bacterial DapB probe, remained very low or absent in monkey all test samples with both pretreatment conditions. PECAM-1

Note: Optimal pretreatment is based on FFPE tissue prepared by CD262, DR5, Cynomolgus Pfizer-Groton. Optimization may be required for samples prepared and 2.5 LS Probe – Mfa-TNFRSF10B TRAILR2, KILLER, 434508 monkey processed differently. TRICK2

2.5 LS Probe – Cl-BBC3 Dog 434378 Control gene selection for tissue qualification 2.5 LS Probe – Cl-CCNE1 Dog 434388 To properly assess the RNA quality of FFPE tissues, it is important to select an appropriate control gene to use. Here we evaluated the 2.5 LS Probe – Cl-CD68 Dog 434398 expression pattern of three housekeeping genes, Polymerase (RNA) 2.5 LS Probe – Cl-Fas Dog 434418 II (DNA directed) polypeptide A (POLR2A), Peptidylprolyl isomerase B (PPIB), and Ubiquitin C (UBC), in different types of tissues from 2.5 LS Probe – Cl-MKI67 Dog 434428 rat, cynomolgus monkey, and dog (Table 2). Our sample selection 2.5 LS Probe – Cl-PECAM1 Dog 434438 covered diverse tissues and organs from each animal (Table 1). 2.5 LS Probe – Cl-TNFRSF10B Dog 434448

2.5 LS Probe – Rn-Bbc3 Rat 434518

2.5 LS Probe – Rn-Ccne1 Rat Puma 434528 Probe Name Species Name Catalog No. Lamp4, Scard1, 2.5 LS Probe – Hs-UBC Homo sapiens 312028 2.5 LS Probe – Rn-Cd68 Rat 402688 gp110 2.5 LS Probe – Hs-PPIB Homo sapiens 313908 2.5 LS Probe – Rn-Fas Rat Tnfrsf6 434738 2.5 LS Probe – Hs-POLR2A Homo sapiens 310458 2.5 LS Probe – Rn-Mki67 Rat 434548 2.5 LS Probe – Cl-UBC Canis lupus 409858 2.5 LS Probe – Rn-Pecam1 Rat CD31, Pecam 315318 2.5 LS Probe – Cl-PPIB Canis lupus 437448 2.5 LS Probe – Rn-Tnfrsf10b Rat 434558 2.5 LS Probe – Cl-Polr2a Canis lupus 310988

2.5 LS Probe – Rn-UBC Rattus norvegicus 312018 TABLE 3. Target specific probes tested in each animal

2.5 LS Probe – Rn-Ppib Rattus norvegicus 313928

2.5 LS Probe – Rn-Polr2a Rattus norvegicus 312488

2.5 LS Negative Control Probe – dapB 312038

TABLE 2. Control probes used in each animal model

4 Application Note Prostate Score= 2+ Epididymis, Score= 3+ Testis, Score= 2+ Ovary, Score= 3+

Stomach, Score= 2-3+ Duodenum, Score= 3+ Jejunum, Score= 3+ Colon, Score= 2+

Kidney, Score= 2+ Bladder, Score= 2+ Lymph node, Score= 2+ Thymus, Score= 1-2+

Esophagus, Score=2+ Skin, Score= 2+ Spinal cord, Score= 2+ Liver, Score+2+

FIGURE 2. Representative images for tissue qualification using the control probe Rn-Ppib in multiple rat tissues. The tissue type and staining score is shown on the bottom right of each image.

Drug Safety Assessment 5 A Hs-UBC Hs-PPIB Hs-POLR2A DapB Cynomolgus monkey

B Hs-POLR2A Hs-PPIB C Hs-UBC Hs-PPIB Retina Heart Muscle Cynomolgus monkey Testis Skeletal muscle Skeletal

D Lymph Node E Duodenum

Rn-Ppib Rn-Ppib Rat

Rn-Polr2a Rn-Polr2a

FIGURE 3. Selection of proper control genes for tissue qualification in RNAscope® ISH assays. (A) Examples of three positive control probes and the negative control probe DapB in cynomolgus monkey FFPE colon tissue. (B) POLR2A and PPIB ISH results in cynomolgus monkey FFPE retina and testis tissues. (C) PPIB and UBC ISH results in cynomolgus monkey FFPE heart and skeletal muscle tissues. (D) Ppib and Polr2a ISH results in rat FFPE lymph node tissue. (E) Ppib and Polr2a ISH results in rat FFPE duodenum tissue.

6 Application Note For rat and cynomolgus monkey, robust and relatively uniform A Colon Ovary PPIB staining was observed with a score of ≥ 2+ in most of the tissues (Figure 2 and Appendix). In the same tissue types, POLR2A ISH usually showed fewer RNA dots, and UBC ISH showed more RNA dots and more clusters of dots compared to PPIB (Figure 3A). Exceptions were observed in the retina and testis, in which the expression of PPIB was lower than that of POLR2A (Figure 3B and CI-UBC Appendix). In the same animal, skeletal and heart muscles showed little to no PPIB ISH signal (Figure 3C). Though PPIB is generally expressed in a uniform manner in the majority of tissues, lymphoid tissues (spleen, lymph node and tonsil) and duodenum showed some non-uniformity in PPIB expression (Figure 3D, 3E and Appendix). In dog tissues, the dot size of PPIB signal was generally smaller than that of POLR2A (Figure 4), due to the limited size of the dog PPIB probe (9 ZZ pairs vs. 14-16 pairs for the rat and human PPIB CI-PPIB probes). Therefore, Cl-POLR2A or Cl-UBC is recommended to be used for tissue qualification, with the requirement of score≥1+ POLR2A( ) or score≥3+ (UBC), respectively. The recommendation for control probe selection in all tissue types and animals used in this study is summarized in Table 4. The full data set of the positive control probe staining patterns is available in the Appendix. Although the majority of the tissues tested showed robust RNA detection, lower signal was observed in the pancreas and alveolar cells from lung tissues, for some unknown reasons. One may CI-POLR2A use UBC as the control gene when detecting high-expressing targets, while detection of medium-to-low expressing RNA may be challenging in these two tissue types if the sample quality is not optimal (Table 4). DapB

FIGURE 4. Selection of proper control probes for tissue qualification in dog tissues for RNAscope® 2.5 LS Assay. ISH results of positive control probes and the negative control probe DapB in dog FFPE colon and ovary tissues.

Drug Safety Assessment 7 Species: Rat and Cynomolgus monkey Detection of specific RNA markers

Tissue type Recommended control probe in FFPE tissues RNAscope® technology can detect virtually any RNA biomarkers Spleen PPIB or POLR2A in situ. Therefore, we wanted to examine the expression of several Lymph node PPIB or POLR2A commonly used targets in preclinical assessment and toxicology studies. Robust, punctate detection of the macrophage marker CD68 Tonsil PPIB or POLR2A was observed in relevant tissues (Figure 5A). Endothelial cells were Thymus PPIB visualized by PECAM1 (CD31) ISH in rat, cynomolgus monkey, and dog (Figure 5B). We used probes for cyclin E1 (CCNE1) and the proliferation Retina POLR2A 3 marker Ki-67 (MKI67) to detect proliferating cells in tissue . The G1-S Spinal cord PPIB phase transition regulator Cyclin E1 (CCNE1) is transcriptionally regulated, while its protein is rather unstable, being rapidly degraded Prostate gland PPIB by the ubiquitin-proteasome system4. Robust ISH detection of CCNE1 Epididymis PPIB was observed in lymphoid tissues and testis, indicating a group of cycling cells (Figure 6A). The proliferation marker Ki-67 (MKI67) was Testis POLR2A also detected in several tissues from the three species examined Ovary PPIB (Figure 6B-C). We also explored targets involved in apoptosis by testing the probes of three p53-regulated pro-apoptotic genes5: Puma Duodenum PPIB (BBC3), CD95 (FAS), and DR5 (TNFRSF10B). In testis, these probes all Jejunum PPIB showed strong detection in a similar region of the tissue (Figure 7).

Colon PPIB

Stomach PPIB

Adrenal gland PPIB

Kidney PPIB

Pancreas* PPIB or UBC

Lung* PPIB or UBC

Urinary bladder PPIB

Esophagus PPIB

Skeletal muscle UBC

Skin PPIB

Liver PPIB

Heart UBC

Species: Dog

Tissue type Recommended control probe

Lung, skeletal and heart muscles UBC

All other dog tissues POLR2A

TABLE 4. Recommendation of control genes for tissue qualification * May use UBC control probe if tissues have low RNA quality, in which case detection of low-medium expressing targets is challenging.

8 Application Note A Rat Cynomolgus monkey Dog CD68

Liver Colon Lymph Node

B Rat Cynomolgus monkey Dog PECAM1

Heart Kidney Liver

FIGURE 5. Detection of cell type specific markers in multiple FFPE tissues by RNAscope® 2.5 LS Assay. (A) ISH results of CD68 in rat liver, cynomolgus monkey colon, and dog lymph node. (B). ISH results of PECAM1 in rat heart, cynomolgus monkey kidney, and dog liver.

Drug Safety Assessment 9 A Lymph Node (Rat) Testis (Rat) CCNE1

B Rat Cynomolgus monkey Dog MK167

Lymph Node Tonsil Testis

C Rat Cynomolgus monkey Dog MK167

FIGURE 6. Detection of Cyclin E1 and the proliferation marker Ki-67 in multiple FFPE tissues by RNAscope® 2.5 LS Assay. (A) ISH results of Ccne1 in rat lymph node and testis. (B) ISH results of MKI67 in rat lymph node, cynomolgus monkey tonsil, and dog testis. (C) ISH results of MKI67 in the duodenum of rat, cynomolgus monkey, and dog.

10 Application Note Conclusions and discussion Bbc3 Rn-Fas In this study, we demonstrate that the fully-automated RNAscope® 2.5 LS Assay is suitable for detecting RNA targets in FFPE tissues from three animal models commonly used in preclinical studies for pharmaceutical development. RNAscope® ISH can be used to detect virtually any drug target candidates and biomarkers for preclinical safety and toxicity assessment. The assay described in this study can be readily set up in any laboratory with required kit reagents and a Leica BOND RX instrument.

Based on the analysis of 25 tissues in 3 animal models, we have Rn-Tnfrsf10b Rn-Polr2a

developed recommendations for control gene selection for tissue Rat Testis qualification for different tissue types (Table 4). We also summarized the threshold of pretreatment needed for different tissue types (Figure 1C-D). The pretreatment guidelines summarized in Figure 1C–D may provide further insight for building tissue microarrays (TMAs), in which multiple tissue types sharing the same assay condition are grouped together. The use of TMAs can be cost- effective when a large number of animal tissues are needed to be analyzed.

While this study was conducted using RNAscope® 2.5 LS Reagent FIGURE 7. Detection of p53-regulated pro-apoptotic molecules in rat testis by Kit-BROWN, similar results can be expected with RNAscope® 2.5 RNAscope® 2.5 LS Assays. RNAscope® ISH results in rat testis using probes Rn-Bbc3, Rn-Fas, Rn-Tnfrsf10b, and control probe Rn-Polr2a. LS Reagent Kit-RED. The same recommendations for positive control genes can also be applied to assays using other RNAscope® platforms, such as RNAscope® 2.5 HD Manual Assays and RNAscope® 2.5 VS Assays. The optimal pretreatment protocol may vary based on RNAscope® platforms, but the general guideline recommending milder pretreatment conditions in certain tissue types may remain valid (Figure 1C and D). For any tissue samples not included in this study, we recommend applying the default pretreatment setting shown in Figure 1C for the initial test, and then optimize as needed to achieve a more robust signal and better morphology if needed.

Appendix containing all data discussed in this study is available at acdbio.com/drugsafetyassessment

Drug Safety Assessment 11 References 1. Cavagnaro J and Silva Lima B. 2015. Regulatory acceptance of animal models of disease to support clinical trials of medicines and advanced therapy medicinal products. European Journal of Pharmacology. 759:51-62.

2. Wang F, Flanagan J, Su N, Wang LC, Bui S, Nielson A, Wu X, Vo HT, Ma XJ, and Luo Y. 2012. RNAscope: A novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. Journal of Molecular Diagnostics, 14(1): 22-29.

3. Camidge DR, Pemberton MN, Growcott JW, Johnstone D, Laud PJ, Foster JR, Randall KJ, and Hughes AM. 2005. Assessing proliferation, cell-cycle arrest and apoptotic end points in human buccal punch biopsies for use as pharmacodynamic biomarkers in drug development. British Journal of , 93(2): 208-215.

4. Hwang HC and Clurman BE. 2005. Cyclin E in normal and neoplastic cell cycles. Oncogene, 24(17): 2776:2786.

5. Chipuk JE and Green DR. 2006. Dissecting p53-dependent apoptosis. Cell Death and Differentiation. 13(6): 994-1002.

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For Research Use Only, not for diagnostic use. RNAscope® and SpotStudio are registered trademarks of Advanced Cell Diagnostics, Inc. in the United States or other countries. All rights reserved. HALO is a registered trademark of Indica Labs, Inc. Leica BOND RX is a California, USA registered trademark of Leica Biosystems. VENTANA and DISCOVERY are trademarks of Roche. ©2015 Advanced Cell Diagnostics, Inc. Doc#: MK 51-035/RevA/Effective date 12/15/2015