In this Issue Feature Article DISCOVERY Yellow Did you know? Technical Tips Thank you! New Products DISCOVERY Teal Beta Testing Begins

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1 ISSUE 02 In this Issue Feature Article Yellow Did you know? Technical Tips Thank you! New Products Teal Beta Testing Begins Yellow Detection Kit By Adrian Murillo, Development Scientist We are very pleased to announce the official launch of the Yellow Kit for research use. After years of inhouse development, Beta Testing, analysis of the Beta Test results, and finally the commercialization of the Yellow Kit, we have created yet another innovative tool for research customers to get the range and specificity they require while performing bright field multiplexing. Yellow detection is unique in the research IHC detection market, as it is a fully stable, opaque and insoluble alkaline phosphatase driven chromogen. Yellow detection is the result of the innovation being performed at Roche Tissue Diagnostics. After optimizing the formulation for maximum stability in a ready-to-use solution, featuring insolubility in alcohol and xylene, the technology was transferred to for in-depth testing, design, and commercialization. The same research and innovation has been responsible for the Purple Detection Kit and other conceptual research use only products that have been or are being Beta Tested. Yellow is a Roche Tissue Diagnostics original, from a novel concept to a groundbreaking, invaluable, and innovative new research multiplexing assay staple. Did you know? Multiplexing Protocol Structure When performing a multiplexing protocol with the Universal Procedure RUO on the Ultra Advanced Staining platform, run antibodies and their corresponding detections separately and sequentially. Quality and clarity of staining could be effected if all antibodies are detected at the same time without knowing how they might respond. Triple stain multiplexing on non-small cell lung carcinoma: CONFIRM Ki67 [30-9] Rabbit Monoclonal Primary Antibody in brown stained with the ChromoMap DAB Kit, Cyotkeratin19 [A53-B/A2.26] in yellow stained with the NP AP Detection system and Yellow Kit, and E-Cadherin (EP700Y) stained with the HQ-HRP Detection System and Purple Kit

2 Yellow - continued Technical Tip Yellow Detection Yellow detection is an ideal second, third or fourth chromogen option for fully automated multiplexing on tissue. It is bright, easy to see, and distinctive. Yellow was chosen as our next research use brightfield offering after the Purple Kit due to its location on the visible light spectrum. With these two detection kits, target adjacency is easy to visually discern. The separation in wavelength between the two chromogens means that they can be quickly and effectively differentiated from one another with digital brightfield hue separation and analysis. With the upcoming Beta release of Teal, it increases the amount of chromogens with which Yellow can co-locate with and thus providing researchers the ultimate flexibility in deciding how best to optimize their particular research assay. To learn more about the technology behind the Yellow detection, you can go here: bioconjchem.5b00652 When multiplexing with Yellow detection, run the antibody you wish to detect with Yellow Kit last or second to last. You should denature any previous secondary antibodies with heat or with the new Ab Denaturation Reagent (Ventana Part Number ). Additional New Product Offerings We are excited to introduce two additional ancillary offerings to the line of Research Use Only products: Ab Denaturation Reagent (Ventana Part Number ) and ISH Diluent (Ventana Part Number ). Both products are new additions for multiplexing on the line of instruments. Ab Denaturation Reagent uses acidic protein elution to remove previously bound primary and secondary antibodies from the tissue for reduced background and non-specific staining while multiplexing. ISH Diluent is specially formulated as both a probe diluent and hybridization solution for geneprotein dual assays. 2 5/9/2017 4:18:27 AM

3 Customer Feedback for the Yellow Kit The Yellow Kit has aided our lab with the ability to obtain strong and definitive staining on both human and murine tissue specimens, resulting in very clear and accurate staining patterns while providing an alternative to the more commonly used DAB staining. It contrasts very well with the Purple chromogen in dual IHC staining, where it has the added benefit of highlighting protein-protein co-localization with a third orange color to demonstrate where there is overlap of the markers used in our studies. Use of such chromogens in dual and multiplex staining yield highly durable staining compared to immunofluor escence and can be conveniently visualized by brightfield imaging. - Hingorani Lab, Fred Hutchinson Cancer Research Center Thank you! Thank you to all of our customers who helped make Yellow detection a success! We began our Beta Test for Yellow in January 2015, and are beyond thrilled to announce that to date, it is our largest and most successful Beta Test. Yellow detection flew off the Beta Shelf, with the largest customer participation base ever! We have received an enormous amount of invaluable feedback that gave us the confidence to go forward, knowing we were offering the best possible addition to our growing brightfield multiplexing portfolio. We strive to give you the best possible research IHC detection solutions on the market, and we could not do it without you. Protein A stained with Purple Kit and Protein B stained with Yellow Kit. Co-localization of the two targets results in a distinctive and bright orange color. Courtesy of Fred Hutchinson Cancer Research Center.

4 Yellow - continued Purple and Yellow Detection Kit appear orange when co-localized. IHC Triplex with cocktail Cytokeratin 5/14 (EP1601Y/LL002) Rabbit and Mouse Monoclonal Primary Antibody (Ventana Medical Systems ) stained with Purple Kit and Yellow Kit, and CONFIRM Ki67 (30-9) antibody stained with ChromoMap DAB Kit. Cytokeratin 5, the rabbit monoclonal antibody, is stained with the HQ-HRP Detection System and Purple Kit. Cytokeratin 14, the mouse monoclonal antibody, is stained with the NP-AP Detection System with Yellow Kit. 4 5/9/2017 4:18:30 AM

5 Protocol Steps for E-Cadherin (EP700Y) and Beta-Catenin (14) Duplex on two different breast cases using Yellow and Purple with NP AP and HQ HRP Detection Systems 1. Deparaffinization 2. CC1 64 minutes 3. Inhibitor, 12 minutes 4. Beta-Catenin at 37 Degrees for 32 minutes 5. Anti-Mouse NP at 37 Degrees for 12 minutes 6. Anti-NP AP for 12 minutes 7. Yellow, 32 minutes 8. Dual Sequence 9. Antibody Denaturation, 95 degrees for 20 minutes 10. E Cadherin, at 37 Degrees for 16 minutes 11. Anti-Rabbit HQ, at 37 Degrees for 12 minutes 12. Anti-HQ HRP, 12 minutes 13. Purple, 36 minutes 14. Counterstain, Hematoxylin II, 12 minutes 15. Post Counterstain, Bluing Reagent, 4 minutes For more information: Visit for more new protocols and lists of necessary reagents.

6 Teal Now in Beta Testing New Beta Testing Reagent Available Teal Detection Kit is s newest chromogenic research detection kit. Teal, like Yellow, is Alkaline Phosphatase driven. Now in Beta Testing, this kit is available for trial under the existing conditions of s Beta Testing program. Teal is an ideal third brightfield chromogen for triplex multiplexing along with Yellow and Purple. Pixel sampling and an assessment in RGB optical density space shows the three chromogens exhibit optimal hue distinction for both easier-to-read multiplex slides and clearer co-localization staining. Teal and Yellow appear green when co-localized. Teal and Yellow Duplex: Estrogen receptor stained with NP AP and Teal and progesterone receptor stained with NP AP and Yellow on breast. products are for research use only. Not for use in diagnostic procedures 6 5/9/2017 4:18:32 AM

7 Word-Search Puzzle D L N K V A E K Y T K M S Q E M T J E L O Y O M N P G H R X R D N N F X N R I V M F U M A U K D O N C W I T I C T W M O Q L U V B L H P A W T B M E A A U D V E T D W J H V N B C O A R R E W S L D G I L N U U U S Z R D N U Y Q H W C A M P C L E A R H I O T T G Q E P X J G P L N O R X G P H I A O K W T W F S C R E A G B Q F R N N P J Y E S E J K Y G X M L Y W T K E D Y E P O C S O R C I M T V C J Y D G R X Q P G S P W F B B P R J M W O L L E Y Y R E V O C S I D W F X F F N A M P L I F I C A T I O N H T D A S G B Y E T P H Q L G U E K V U B K S N I E T O R P E N E G U U T M X G K Q Q I L V M I C R O R N A G E L P R U P Y R E V O C S I D R K C Find and circle these words: AMPCLEAR AMPLIFICATION DENATURATION PURPLE YELLOW FITC GENEPROTEIN MICRORNA MICROSCOPE MRNA MULTIPLEX RHODAMINE Solution for Word-Search Puzzle D L N K V A E K Y T K M S Q E M T J E L O Y O M N P G H R X R D N N F X N R I V M F U M A U K D O N C W I T I C T W M O Q L U V B L H P A W T B M E A A U D V E T D W J H V N B C O A R R E W S L D G I L N U U U S Z R D N U Y Q H W C A M P C L E A R H I O T T G Q E P X J G P L N O R X G P H I A O K W T W F S C R E A G B Q F R N N P J Y E S E J K Y G X M L Y W T K E D Y E P O C S O R C I M T V C J Y D G R X Q P G S P W F B B P R J M W O L L E Y Y R E V O C S I D W F X F F N A M P L I F I C A T I O N H T D A S G B Y E T P H Q L G U E K V U B K S N I E T O R P E N E G U U T M X G K Q Q I L V M I C R O R N A G E L P R U P Y R E V O C S I D R K C

8 How do you become a Beta Tester? Beta Products Participation in the Beta Test program is open to all VENTANA ULTRA users. Requirements: ULTRA instrument users with the ULTRA Universal Procedure Willingness to work with un-released beta reagents Provide feedback on beta reagent performance within a 90 day time frame Passion for innovation and desire to have an impact our product pipeline What to expect: ONE (1) 50 test kit of beta reagent Protocol training for beta product by training class, or webinar 2 month dating to allow for testing 90 day turn around on performance feedback Beta reagents will not be used in project optimization In order to remain in the program you must provide feedback **Some of these products do not become products based off the customer feedback we receive and we do ask customers not to start projects with these due to the time it does take to create a commercial product if the product is well received. Roche Diagnostics (Schweiz) AG Industriestrasse 7 CH-6343 Rotkreuz Schweiz Tel: +41 (0) Fax: +41 (0) Ventana Medical Systems, Inc. All trademarks mentioned enjoy legal protection. VENTANA and are trademarks of Roche. All other trademarks are the property of their respective owners. 6253A RTDPC-DIS-0034