C-MYC/CEP8

Product Name

MYC(8q24) gene amplification probe reagent.

Package Specifications

10 Tests/box.

Intended use

The reagent carries out in situ hybridization staining on the basis of routine staining to provide doctors with auxiliary information for diagnosis. The test results are only for clinical reference and should not be used as the only basis for clinical diagnosis. Clinicians should comprehensively judge the test results in combination with the patient’s condition, drug indications, treatment response and other laboratory test indicators.

Detection principle

Fluorescence in situ hybridization is a technique for directly observing specific nucleic acids in cells. According to the principle of base complementary pairing, the specific probe is complementary to the target sequence in the cell. Due to the fluorescence of the probe,the gene state of the hybrid probe and the target sequence can be clearly observed under the fluorescence microscope under the appropriate excitation light.

Product Composition

The kit consists of C-MYC/CEP8 dual color probe as shown in Table 1.

Storage conditions & Validity

Keep sealed away from light at -20^oC±5^oC. 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^oC in dark. For long-term preservation after opening, keep the lid sealed at -20^oC±5^oC away from light. The kit is transported below 0^oC.

Applicable Instruments

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

Sample Requirements

1. Applicable specimens’ types: Surgical excision or paraffin-embedded biopsy specimens.
2. The tissue should be fixed with 4% neutral formaldehyde fixation solution within 1 hour. After tissue fixation, it should be regularly dehydrated and embedded in paraffin.

Test method

1. Pretreatment
   1. Baking: Slides heating at 80°C for 30min or 65°C for 2h or overnight.
   2. Dewaxing: According to the customer laboratory protocol (Commonly with Xylene for 15min).
   3. 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.
   4. Permeation: Immerse the slides in deionized water at 100°C and boil continuously for 20-40 minutes (Conventional 20min). Remove the excess of water on the slides by air-drying.
   5. 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.
   6. Washing: Wash with 2xSSC at room temperature for 5 minutes.
   7. 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 22mmx22mm 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. (Preparation of 0.3% NP-40/0.4xSSC: For 1L preparation, take 3mL NP-40 and 20mL 20xSSC, dissolve fully, mix well, and use 1M NaOH to adjust the pH to 7.2).
   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. Dyeing
   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 sections under a fluorescence microscope and the cells area is first confirmed under a low magnification objective (10x).Under magnification objective (40x) a uniform cell distribution is observed.Then the nucleus size uniformity, nuclear boundary integrity, DAPI staining uniformity, no nuclei overlapping, and cells clear signal are observed in the high magnification objective (60x, 100x).

Precautions

1. The results of this kit can be affected by variousfactors of the sample itself, as well as being limited by the temperature and hybridization time, the operating environment and limitations of current molecular biology techniques, which may lead to erroneous results.
2. The user must understand the limitations of potential errors and inaccuracies that may exist in the testing process.