ETV6

Product Name

ETV6 gene break apart probe reagent

Package Specifications

10 Tests/box

Product Introduction

This kit uses orange fluorescein and green fluorescein to label ETV6 probe. ETV6 dual color probe can be combined with the target detection site by in situ hybridization.

Product Content

This kit consists of ETV6 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 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

Cell:
1. Take 1-3ml of heparin sodium anticoagulant bone marrow cells.
2. Sample preservation: Fresh bone marrow specimen without fixation (preserved at 2-8^oC for no more than 24 hours). After fixation, the cell suspension can be preserved at -20±5^oC for no more than 12 months; the prepared cell slide can be preserved at -20±5^oC for no more than 1 month. When the storage temperature of the sample is too high or too low, the cell suspension is volatilized excessively or polluted,the sample cannot be used for detection.

Tissue:
1. Applicable specimen types: Paraffin-embedded specimensfrom surgical excision or biopsy.
2. The tissue should be fixed with 4% neutral formaldehyde solution within 1 hour after isolation. After tissue fixation, it is routinely dehydrated and embedded in paraffin.

Test method

1. Related reagents

• 20×SSC, pH 5.3±0.2
  Weigh 176g of sodium chloride and 88g of sodium citrate, dissolve in 800mL of deionized water, adjust the pH to 5.3±0.2 at room temperature, and complete to 1 L with deionized water. High-pressure steam sterilization, stored at 2-8oC,the solution shelf life is of 6 months. Discard if the reagent appears cloudy (turbid) or contaminated.
• 2×SSC, pH 7.0±0.2
  Take 100mL of the above 20xSSC, dilute with 800mL deionized water, mix, adjust the pH to 7.0±0.2 at room temperature, complete to 1L with deionized water, stored at 2-8oC, the shelf life is of 6 months. Discard if the reagent appears cloudy (turbid) or contaminated.
• Ethanol Solution: 70% ethanol, 85% ethanol
  Dilute 700ml, 850ml of ethanol with deionized water to 1L. The shelf life is of 6 months. Discard if the reagent appears cloudy (turbid) or contaminated.
• 0.3% NP-40/0.4xSSC solution, pH 7.0-7.5
  Take 0.6mL NP-40 and 4mL 20×SSC, add 150mL deionized water, mix, adjust the pH to 7.0-7.5 at room temperature, with deionized water complete to a volume of 200mL. Stored at 2-8oC, the shelf life is of 6 months. Discard if the reagent appears cloudy (turbid) or contaminated.
• Fixation solution (methanol: glacial acetic acid = 3:1)
  Prepare a ready to use fixation solution by mixing thoroughly 30ml of methanol and 10ml of glacial acetic acid.
• 0.075M KCl solution
  Weigh 2.8g of potassium chloride, dissolve in 400mL of deionized water and complete to 500mL with deionized water. Stored at room temperature, the solution shelf life is of 6 months. Discard if the reagent appears cloudy (turbid) or contaminated.
• Diamidinyl phenylindole (DAPI) counterstain
  Use commercially available anti-quenching DAPI counterstain.

Cells sample:
2. Sample processing before hybridization
Cells sample:

1. Sample collection: Take 1-3 mL of heparin sodium anticoagulated bone marrow cell sample.
2. Cell harvest: Pipet uncultured bone marrow cells or cultured bone marrow cell samples into a 15mL conical centrifuge tube, centrifuge at 500g for 5 minutes, carefully aspirate and discard the supernatant, and leave about 500μL of residual liquid to resuspend the cells.
3. Cell washing: Add 5mL of 1×PBS solution by pipetting to mix and resuspend the cell pellet, centrifuge at 500g for 5min, carefully aspirate and discard the supernatant, keep about 500μL of residual liquid to resuspend the cells; repeat once.
4. Cell permeation: Add 10mL hypotonic solution to each tube (pre-warmed at 37°C bath) and place at 37°C water bath hypotonic for 20min.
5. Cell pre-fixation: Add 1mL (10% volume) of fixative to the cell suspension after permeation to pre-fix the cells, gently pipette to mix,and immediately centrifuge at 500g for 5min, and remove the supernatant, keep about 500μL of residual liquid to resuspend the cells.
6. Cell fixation: Slowly add 10 mL of fixative to the cell suspension, put at room temperature for 10 min to fix the cells. Centrifuge at 500g for 5 min, and keep about 500μL of residual liquid to resuspend the cells; repeat once (the cells can also be fixed multiple times until the cells precipitate and wash out).
7. Preparation of cell suspension: After the last cell fixation and centrifugation, aspirate the supernatant and add an appropriate amount of fixative to prepare the cell suspension with the appropriate concentration.
8. Slide preparation: 3-10μL cell suspension was dropped onto the slide and aged at 56 °C for 0.5h.
9. Pretreatment: the slides were rinsed twice in 2×SSC solution at room temperature for 5min each time.
10. Dehydration: the cell drops were placed in 70% ethanol, 85% ethanol and 100% ethanol for 2 minutes respectively and then dried naturally.

Tissue sample:
Baking: Slides heating at 80oC for 30min or 65oC 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 100oC 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 0.5%) 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.

3. Denaturation and Hybridization
The following operations need to be carried out in the darkroom.
Cell sample:

1. Take out the probe, leave it at room temperature for 5min, turn it upside down with force, mix it well, and then centrifuge it for a short time (no vortex instrument vibration). Take 10μL of it and drop it into the cell drop hybridization area, immediately cover the cover glass of 22mm × 22mm. The probe should be evenly expanded under the cover glass without bubbles, and seal the edge with rubber glue (the edge must be completely sealed to prevent the dry piece from affecting the test results in the hybridization process).
2. The cell drops were placed on the hybridizer and denatured at 88oC for 2min (the hybridizershould be preheated to 88oC) and hybridized at 45oC for 2 to 16 hours.

Tissue samples:

1. Take out the probe, let it stand at room temperature for 5min, turn it upside down with force, fully mix the probe, and then centrifuge briefly (vortex instrument oscillation is prohibited), take 10μL was dropped on the hybridization area of cell drops and immediately covered with 22mm×22mm cover glass, the probe shall be evenly expanded under the cover glass without bubbles, and the edge shall be sealed with rubber glue (the edge must be completely sealed to prevent the dry piece from affecting the test results during hybridization).
2. Put the tissue sections on the hybridizer, CO denature at 85°C for 5min (the hybridizer should be preheated to 85°C in advance), and hybridize at 42°C for 2-16h.

4. Washing
The following operations need to be carried out in the darkroom.

1. Carefully remove the sealing glue around the cover glass with tweezers to avoid sticking or moving the cover glass, immerse the sample in 2xSSC for about 5S, take it out, gently push a corner of the cover glassto the edge of the slide with tweezers, and gently remove the cover glass with tweezers;
2. Place the sample at 2xSSC room temperature for 1 min;
3. Take out the slides and immerse in a preheated at 68°C 0.3% NP-40/0.4xSSC (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) solution and wash for 2min.
4. Take out the sample and immerse it in deionized water preheated at 37^oC in advance for 1min; dry it naturally in the dark environment.

5. 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.

6.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 are observed.

Interpretation of common signal types

ETV6 gene site 5 signal

ETV6 gene site 3 signal

Negative: 2 fusion

Positive: 1 orange 1 green 1 fusion

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 off.