Immunohistochemistry. Services & Products. Indivumed GmbH Falkenried 88, Bldg. D Hamburg Germany

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1 Services & Products Indivumed GmbH Falkenried 88, Bldg. D Hamburg Germany Phone Europe: Fax Europe: Phone USA: info@indivumed.com

2 Immunohistochemistry custom service Staining on FFPE and frozen tissue High quality tissue comprehensive clinical data Protocol development Detection of phosphoproteins Several established antibodies, e.g., anti-p-mtor, anti-p-mapk Activated / non activated form IHC using an automated staining platform Ventana Discovery XT Highly controlled and standardized conditions Detection of apoptosis Several established antibodies, e.g., anti-caspase-3, anti-bcl-2, anti-p53 Documentation and reporting Evaluation by pathologist Patient and clinical data Digital images Tissue BioChips (FFPE and Frozen) Newly developed product Standardized spots (squares) Ideal for target validation and antibody screening HER-2/neu status in breast cancer Clone 4B5, FDA approved Routine staining of all breast cancer tissues FISH and SISH testing For details, please klick on image

3 Indivumeds immunohistochemistry custom service The complete immunohistochemistry (IHC) service includes: Standardized staining with commercially available antibodies or research antibodies (antibodies can be provided by the customer) via Ventana Technology Protocol development and staining on FFPE or Fresh Frozen Tissue Optimization of staining protocols e.g., with respect to antigen retrieval conditions (detergent, enzymatic or microwave methods), antibody concentration and incubation time High quality digital images (Mirax Scan from Zeiss) Comprehensive report of the findings and microscopic images For target validation and antibody screening Indivumed provides high quality tissue (FFPE and Fresh Frozen) with documented ischemia time and comprehensive clinical data, H&E staining and quality control for each tissue block. Staining includes positive and negative controls Stainings either with immunoperoxidase or alkaline phosphatase methods (DAB Map and ultra Map detection kits). Interpretation of immunostaining results by our pathologists 3

4 List of established antibodies Antibody Clone Tissue Type ACLY poly FFPE AKT (pan) C67E7 FFPE ATM SYR6Da FFPE Bcl-2 (Oncoprotein) 124 FFPE / FF BrdU IIB5 FFPE / FF BrdU BU1/75 FFPE Calcitonin poly FFPE CAR RmcB FF Caspase 3 poly FFPE Caspase-3, cleaved 5A1E FFPE CD31 SZ31 FFPE Cyclin B1 poly FFPE Cyclin D1 SP4 FFPE ß-Catenin poly FFPE Cytokeratin (pan) KL1 FFPE / FF EGFR FFPE EGFR 3C6 FFPE EGFR (PharmaDX) 2-18C9 FFPE Estrogen Receptor Sp1 FFPE / FF FAK poly FFPE FAS C20G5 FFPE FHIT poly FFPE Folate Receptor-Alpha BN3.2 FFPE GLUT1 mono FFPE gp96 poly FFPE GRP94 poly FFPE Her-2/neu (Pathway) 4B5 FFPE / FF Her3 / ErbB-3 poly FFPE Hekokinase II C64G5 FFPE HSP90 C45G5 FFPE KGF / FGF7 poly FFPE Ki67 BGX-297 FFPE / FF Ki67 SP6 FFPE Antibody Clone Tissue Type LDHA C4B5 FFPE MCT1 poly FFPE MCT4 poly FFPE mtor/frap Y391 FFPE mtor 7C10 FFPE p16 (INK4) G FFPE p27 (Kip1) Y236 FFPE P27 (Kip1) EPFHCR16 FFPE p53 Do7 FFPE / FF p63 4A4 FFPE p-akt (ser473) 736E11 FFPE p-akt (ser473) D9E FPPE p-egfr (Tyr1173) E124 FFPE p-fak (Tyr397) poly FFPE p-gsk3 beta EPR2286Y FFPE p-her-3 (Tyr1289) 21D3 FFPE p-histone H3 E173 FFPE p-mapk 20G11 FFPE p-mtor (Ser2448) 49F9 FFPE / FF p-p27kip1 (Ser10) EP233(2)Y FFPE p-p70 S6 Kinase (pt389) E175 FFPE p-paxillin (Tyr31) E228 FFPE p-tyrosine p-tyr-100 FFPE / FF p-stat5 (Tyr694) E209 FFPE PDK1 (Pyruvate dehydrogenase kinase isozyme 1) poly FFPE PDK1 (3-Phosphoinositdedependent protein kinase-1) Y336 FFPE Progesterone Receptor SP2 FFPE / FF PTEN 138G6 FFPE STK11 poly FFPE VEGFR-2 55B11 FFPE Vimentin V9 FFPE / FF FFPE = Formalin Fixed Paraffin Embedded FF = Fresh Frozen 03/2011 4

5 IHC using an automated staining platform Indivumed has established an IHC service platform for analyzing various biomarkers and drug targets in the area of oncology. IHC service is performed on the Ventana Discovery XT Automated Staining Platform. The system runs under highly controlled and standardized conditions. 5

6 IHC automated staining platform The Ventana System enables: Flexible protocol development Automated slide preparation with respect to baking, deparaffinization, cell conditioning and staining Precise individual slide heating by a patented ThermoPad slide platform High reproducibility with ready-to-use reagents (Ventana) Simultaenous processing for up to 30 slides IHC and ISH procedures 6

7 Documentation and reporting The Indivumed IHC custom service includes a comprehensive report with the following information: Detailed staining protocol for each specific antibody using the Ventana Discovery XT Patient and clinical data (when tissue sections are provided by Indivumed) Description ot the results and summary statement (done by staff pathologist) Colored microscopic images in different magnifications (Mirax Scan) On request, digital images can be provided Digital Images are performed with the Mirax Scan from Zeiss. The Mirax Digital Slide Scanner is the system solution for digital pathology, which allows high quality evaluation from any place at any time. In addition, pictures of brilliant quality can be made from those digital scans. 7

8 HER-2/neu status in breast cancer Anti-HER-2/neu Immunoperoxidase staining of formalin fixed, paraffin embedded human breast - ductal carcinoma shows strong membranous positivity in epithelial cells At Indivumed, all breast cancer tissues are stained on a routine basis for HER-2 expression to provide customers with exact results for each tissue block. BildunterschriftUsciliqu amconullum ing ero odolorp eraest HER-2 staining is performed on the Ventana Discovery XT platform using Ventana s PATHWAY anti-her-2/neu (4B5) antibody. Anti-HER-2/neu is a rabbit monoclonal primary antibody (clone 4B5) directed against the internal domain of the c-erbb-2 oncoprotein (HER2). This antibody has been optimally diluted for use with Ventana detection kits (DAB Map) and reveals little signal to noise ratio. Her-2/neu (4B5) is FDA approved and intended for in vitro diagnostic (IVD) use. On request, HER-2 (2+) cases can be validated by FISH and SISH technology. 8

9 Detection of phosphoproteins Established protocols for phosphoproteins, e.g.: p-akt / AKT (FFPE) p-egfr / EGFR (FFPE) p-fak (FFPE) p-mapk (FFPE) p-mtor p-tyrosine (FFPE / FF) (FFPE / FF) Protein phosphorylation is one of the key post-translational modifications regulating protein function and appears to be most consistent with their biological activities. In cancer cells, prolonged phosphorylation leads to constitutive activation of signal transduction pathways. Indivumed has estalished protocols for a panel of different phosphoproteins (non activated / activated form). 9

10 Detection of phosphoproteins Example I: p-mtor Anti-p-mTOR (Ser2448) Immunoperoxidase staining of formalin fixed, paraffin embedded human colon - adenocarcinoma shows membranous and cytoplasmic positivity in epithelial cells BildunterschriftUsciliqu amconullum ing ero odolorp eraest 10

11 Detection of phosphoproteins Example II: p-mapk Anti-p-p44/42 MAPK (Thr202/Tyr204) Immunoperoxidase staining of formalin fixed, paraffin embedded human colon - adenocarcinoma shows nuclear and cytoplasmic positivity in epithelial cells BildunterschriftUsciliqu amconullum ing ero odolorp eraest 11

12 Detection of apoptosis Anti-Caspase 3 (Cleaved) Immunoperoxidase staining of formalin fixed, paraffin embedded human lung - adenocarcinoma shows nuclear and cytoplasmic positivity in epithelial cells Indivumed has estalished several protocols for apoptosis related proteins, e.g. caspase-3, Bcl-2, Bax, p53. BildunterschriftUsciliqu amconullum ing ero odolorp eraest Apoptosis, also known as programmed cell death, is a key biochemical process whereby an organism regulates cell numbers and developmental pathways. This process has been shown to be a key factor in many diseases, including cancer, and is a target for research and drug discovery. Diverse groups of molecules are involved in the apoptosis pathway. Especially caspase-3 has been identified as being a key mediator of apoptosis of mammalian cells. 12

13 Tissue BioChips (FFPE and Frozen) Tissue BioChips are a newly developed product at Indivumed providing superior quality compared to conventional Tissue Microarrays: Defined content in all spots by scanning of tissue slices and individual selection of areas 2x2 mm standardized spots (squares) No loss of tissue due to incomplete cores Available from fresh frozen tissue as well as FFPE tissue Integration of spotted cells possible (e.g., staining control) Tissue BioChips are ideal for target validation and antibody screening and can be stained automatically on the Ventana Discovery XT using established protocols. 13