ZytoLight SPEC HER2/CEN 17 Dual Color Kit - Manual -

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1 - Manual - Heat Pretreatment Proteolysis Denaturation / Hybridization ZytoVision GmbH

2 5.1 Preparatory Steps Day 1 Preparation of two ethanol series (70%, 90%, 100%) Heat Pretreatment Solution Citric (PT1): Heat in a covered staining jar standing in a boiling water bath to 98 C. Place filled staining jar into a cold water bath and heat water bath to 98 C temperature inside staining jar should be approx. 96 C. Pepsin Solution (ES1): Bring to room temperature. Wash Buffer SSC (WB1): Bring to room temperature ZytoVision GmbH

3 5.2 Pretreatment (Dewax/Proteolysis) (day 1) 1. Incubate 10 min at 70 C 2. Incubate 2x 10 min in xylene 3. Incubate in 100%, 100%, 90%, 70% ethanol, each for 5 min Alternatively, dewaxing protocols routinely used in immunohistochemistry procedures, e.g. 2x 15 min xylene, 2x 5 min 100% ethanol, 2x 5 min 96% ethanol, 1x 5 min 70% ethanol, can be used. 4. Wash 2x 2 min in water 5. Incubate for 15 min in pre-heated Heat Pretreatment Solution Citric (PT1) We recommend not to use more than six slides per staining jar as each slide will decrease the temperature! 6. Wash 2x 2 min in water, and drain off or blot off the water (to avoid dilution of the pepsin solution also air drying is possible!) STOP possible (store slides in tap water for approx. 3-4 h or air-dried ) Heat Pretreatment ZytoVision GmbH

4 7. Apply Pepsin Solution (ES1) and incubate for approx. 10 min at 37 C in a humidity chamber Proteolysis Incubation time depends on: nature of tissue duration of fixing type of fixative thickness of sections We recommend an incubation time of min for tissue samples. 8. Wash 5 min in Wash Buffer SSC (WB1) Wash 1 min in water 9. Dehydration: in 70%, 90%, 100% ethanol, each for 1 min Air dry sections STOP possible (for several months) Wash ZytoVision GmbH

5 5.3 Denaturation and Hybridization (day 1) 1. Vortex the ZytoLight SPEC HER2/CEN 17 Dual Color Probe (PL8) and pipette 10 µl each onto individual samples A gentle warming of the probe, as well as using a pipette tip, which has been cut off to increase the size of the opening, can make the pipetting process easier. Apply Probe Avoid long exposure of the probe to light! 2. Cover the samples with a coverslip (avoid trapping bubbles) Seal the coverslip, e.g. with rubber cement It is essential that the tissue/cell samples do not dry out during the hybridization step. 3. Denature the slides at 75 C (±2 C) for 10 min on a hot plate Lowering the denaturation temperature to 65 C is possible and might influence signal size and pepsin digestion time! 4. Transfer the slides to a humidity chamber and hybridize overnight at 37 C (e.g. in a hybridization oven or Hybrite) Denature & Hybridize ZytoVision GmbH

6 - Manual - Washing Detection ZytoVision GmbH

7 5.1 Preparatory Steps Day 2 Prepare 1x Wash Buffer A by diluting 25x Wash Buffer A (WB2). Prepare three staining jars with Wash Buffer A and prewarm to 37 C Bring to room temperature: DAPI/Antifade-Solution (MT1) ZytoVision GmbH

8 Post-Hybridization and Detection (day 2) 1. Carefully remove the rubber cement or glue 2. Remove the coverslip by submerging in 1x Wash Buffer A at 37 C for 1-3 min 3. Wash 2x 5 min in 1x Wash Buffer A at 37 C The 1x Wash Buffer A should be pre-warmed. Check with a thermometer if necessary. 4. Incubate in 70%, 90%, and 100% ethanol, each for 1 min Air dry the samples protected from light If, at evaluation, slides appear foggy or cloudy - especially when using the orange filter - consider to prepare a fresh ethanol series. Some kinds of permanent markers (Edding) tend to produce high fluorescence background. Wash ZytoVision GmbH

9 5. Apply DAPI/Antifade-Solution (MT1) Cover the sample with a coverslip avoiding trapped bubbles Incubate for 15 min at RT in the dark Using a pipette tip which has been cut off to increase the size of the opening, can make the pipetting process easier. Avoid long exposure to light. Apply DAPI/ Antifade ZytoVision GmbH

10 6. Carefully remove excess DAPI/Antifade-Solution (MT1) by gently pressing the slide between filter papers 7. Evaluation of the sample material is carried out by fluorescence microscopy Use fluorescence microscopy approved oil only! Store slides at +4 C to -20 C! ZytoVision GmbH

11 Required filter sets: HER2 (ZyGreen ): CEN 17 (ZyOrange ): excitation at 503 nm, emission at 528 nm (similar to FITC) excitation at 547 nm, emission at 572 nm (similar to Rhodamine) ZytoVision GmbH