C-MET/CEP7

Product Name

MET gene probe reagent.

Package Specifications

10 Tests/box.

Product Introduction

The kit uses orange fluorescein-labeled C-MET probe and green fluorescein-labeled CEP7 probe to bind C-MET/CEP7 probe to the target detection site by in situ hybridization.

Product Composition

The kit consists of C-MET/ CEP7 dual-color probes, as shown in Table 1.

Storage conditions & Validity

Keep sealed away from light at -20°C±5°C. The product is valid for 12 months. Avoid unnecessary repeated freezing and thawing that should not exceed 10 times. After opening, within 24 hours for short-term preservation, keep sealed at 2-8°C in dark. For long-term preservation after opening, keep the lid sealed at -20°C±5°C away from light. The kit is transported below 0°C.

Applicable Instruments

Fluorescence microscopy imaging system includes fluorescence microscopy and filter sets for DAPI (367/452), Green (495/517), and Orange (547/565).

Sample Requirements

1. Applicable specimen type: paraffin embedded specimen of surgical resection or biopsy tissue.
2. The tissue should be fixed with 4% neutral formaldehyde fixation solution within 1 hour after in vitro. After tissue fixation, it should be regularly dehydrated and embedded in paraffin.

Related Reagents

1. Pretreatment
   Baking: Slides heating at 80^oC for 30min or 65^oC for 2h or overnight.
   Dewaxing: According to the customer laboratory protocol (Commonly with Xylene for 15min).
   Hydration: Take out the slides and put them respectively into 100%, 85%, and 70% EtOH at room temperature for 3 minutes each. Take out the slides, and immerse them in deionized water for 3 minutes. Remove the excess of water on the slides by air-drying.
   Permeation: Immerse the slides in deionized water at 100^oC and boil continuously for 20-40 minutes (Conventional 20min). Remove the excess of water on the slides by air-drying.
   Digestion: Protease enzymic digestion at 37°C for 10-40 minutes. Mix the protease work buffer (50mmol HCl) and the 10x protease solution (Pepsin concentration 5%) in a proportion of 9:1 to prepare the enzymatic digestion solution.
   Washing: Wash with 2xSSC at room temperature for 5 minutes.
   Dehydration: Take out the slides and dehydrate in 70%, 85%, and 100% gradient ethanol at room temperature for 2 minutes each time. Remove the excess of EtOH solution on the slides by air-drying.
2. Denaturation Hybridization
   The following operations should be performed in a darkroom.
   1. Take the probe at static room temperature for 5 minutes. Briefly centrifuge (1-2s) after manually mixing the probe (do not use vortex/swirl or shaker instrument/oscillator). Take 10μl droplet in the cell and drop in the hybridization zone, immediately cover 22mm x 22mm glass slide area; spread evenly without bubbles the probe under the glass slide covered area and seal edges with rubber (edge sealing must be thorough to prevent dry film from affecting the test results during hybridization).
   2. Place the glass slide in the hybridization instrument, denature at 85°C for 5 minutes (the hybridizer should be preheated to 85°C) and hybridize at 42°C for 2 to 16 hours.
3. Washing
   The following operations should be performed in a darkroom.
   1. Take out the hybridized glass slides, remove the rubber on the coverslip and immediately immerse the slides in a 2xSSC solution for 5 seconds and remove the coverslip.
   2. Place the slides in a 2×SSC at room temperature for 1 min.
   3. Take out the slides and immerse in a preheated at 68°C 0.3% NP-40/0.4xSSC solution and wash for 2 min.
   4. Remove the slides and immerse in a 37°C preheated deionized water, wash for 1 min, and dry the slides naturally in the dark.
4. Counterstaining
   The following operations should be performed in a darkroom.
   10μl DAPI compound dye is dropped in the hybridization area of the glass slide and immediately covered. The suitable filter is selected for glass slide observation under the fluorescence microscope.
5. FISH results observation
   Place the stained slides under a fluorescence microscope and confirm the cells area under a low magnification objective (10x). Under magnification objective (40x) a uniform cells distribution is observed. Then the nuclei FISH results are observed under the high magnification objective (100x).

Common Signal Type Interpretation

C-MET

CEP7

Negative: 2 orange 2 green

Positive: n Orange 2 Green(n≥4))

Precautions

1. The results of this kit will be affected by various factors of the sample itself, but also limited by hybridization temperature and time, operating environment, and the limitations of current molecular biology technology, which may lead to wrong results.
2. Users must understand the potential errors and accuracy limitations that may exist in the detection process.
3. All chemicals are potentially dangerous. Avoid direct contact. Used kits are clinical waste and should be properly disposed of.

Manual Approval date & Revision date

V1. 0: Approval date: September 3, 2018.
V1. 1: Revision date: December 7, 2021.