CEN 17 Dual Color Kit - Manual -

ZytoLight ® SPEC HER2/CEN 17 Dual Color Kit - Manual Heat Pretreatment Proteolysis Denaturation / Hybridization

Semua bahan yang dibutuhkan dalam protocol ini, dapat dipesan di: PT. Biozatix Indonesia Website: www.biozatix.com Email: [email protected]

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© 2010 ZytoVision GmbH

ZytoLight ® SPEC HER2/CEN 17 Dual Color Kit 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.


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ZytoLight ® SPEC HER2/CEN 17 Dual Color Kit 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)

Heat Pretreatment

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 ) 3


ZytoLight ® SPEC HER2/CEN 17 Dual Color Kit 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 10-30 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 Semua bahan yang dibutuhkan dalam STOP possible (for several months) 4

Wash

protocol ini, dapat dipesan di: PT. Biozatix Indonesia Website: www.biozatix.com Email: [email protected]


ZytoLight ® SPEC HER2/CEN 17 Dual Color Kit 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.

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

Apply Probe


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!

Denature & Hybridize

4. Transfer the slides to a humidity chamber and hybridize overnight at 37°C (e.g. in a hybridization oven or Hybrite) 5


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


Washing Detection 1


ZytoLight ® SPEC HER2/CEN 17 Dual Color Kit 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)


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ZytoLight ® SPEC HER2/CEN 17 Dual Color Kit Post-Hybridization and Detection (day 2) 1. 2. 3.


Carefully remove the rubber cement or glue Remove the coverslip by submerging in 1x Wash Buffer A at 37°C for 1-3 min Wash 2x 5 min in 1x Wash Buffer A at 37°C

Wash

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.

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ZytoLight ® SPEC HER2/CEN 17 Dual Color Kit 5.

Apply DAPI/Antifade-Solution (MT1) Cover the sample with a coverslip avoiding trapped bubbles Incubate for 15 min at RT in the dark

Apply DAPI/ Antifade

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.


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ZytoLight ® SPEC HER2/CEN 17 Dual Color Kit 6. 7.

Carefully remove excess DAPI/Antifade-Solution (MT1) by gently pressing the slide between filter papers 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!


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ZytoLight ® SPEC HER2/CEN 17 Dual Color Kit 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)


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CEN 17 Dual Color Kit - Manual -

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