FISH BIO ROS1 PROBE

Product Name

6q probe reagent.

Package Specifications

10 Tests/box

Product Introduction

The kit adopts 6q probes labeled with orange fluorescein and green fluorescein. The gene rearrangement of 6q can be detected by in situ hybridization.

Product Main Components

The kit consists of 6q dual color probe, 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 under 0°C.

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 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 tissue fixation, it should be regularly dehydrated and embedded in paraffin.

Instructions

1. Hybridization pretreatment
   • Baking: Slides heating at 80°C for 30min or 65°C 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°C 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 and Hybridization
   The following operations should be performed in a darkroom.
   1. Take out the probe put at room temperature for 5min. Mix and centrifuge briefly. 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 slides 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 place the slides into 2xSSC for 5 seconds, and gently 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 2min. (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 1min and dry the slides naturally in the dark.
4. Counterstaining
   The following operations should be performed in a darkroom.
   Dip 10μL of DAPI counterstain into the hybridization area of the glass slides, immediately cover and then use the appropriate filter to observe the sections under the fluorescence microscope.
5. FISH results observation
   Place the counterstained film under the fluorescence microscope, and first put it under the low-power objective lens (10x) confirm the cell area under the microscope; Go to 40x under the objective lens, find a position where the cells are evenly distributed; Then in the high- power objective (100x) the FISH results of nuclei were observed.

Common Signal Type Interpretation

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 hazardous wastes and should be properly disposed of.