CEP8

Product Name

Chromosome 8 centromere probe reagent.

Package Specifications

10 Tests/box

Product Introduction

The kit uses orange fluorescein-labeled CEP8 to bind CEP8 probe to the target detection site by in situ hybridization.

Product Composition

The kit consists of CEP8 orange 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 thatshould 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 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: Unfixed fresh bone marrow specimen (stored at 2-8°C for no more than 24 hours).
2. Sample collection: Take 1-3ml of bone marrow cells anticoagulated with heparin sodium.
3. Sample preservation: After fixation, the cell suspension should be stored at -20±5°C for no more than 12 months. The prepared cell slides can be stored at -20±5°C for no more than 1 month.If the sample storage temperature is too high or too low,If the cell suspension is excessively volatilized or contaminated during storage,The sample will not be suitable for detection.

Related Reagents

The following reagents are required for the experiment but not provided in this kit

1. 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 1L with deionized water. Sterilize using high-pressure steam and store at 2-8°C with a shelf life of 6 months. Discard if the reagent appears cloudy (turbid) or contaminated.
2. 2×SSC, pH 7.0±0.2:
   Take 100mL of the prepared 20×SSC, dilute with 800mL of deionized water, mix, adjust the pH to 7.0±0.2 at room temperature, and complete to 1L with deionized water. Store at 2-8°C with a shelf life of 6 months. Discard if the reagent appears cloudy (turbid) or contaminated.
3. Ethanol Solution:
   Prepare 70% ethanol and 85% ethanol by diluting 700mL and 850mL of ethanol with deionized water to 1L. The shelf life is 6 months. Discard if the reagent appears cloudy (turbid) or contaminated.
4. 0.3% NP-40/0.4×SSC solution, pH 7.0-7.5:
   Take 0.6mL NP-40 and 4mL 20×SSC, add 150mL of deionized water, mix, adjust the pH to 7.0-7.5 at room temperature, and complete to 200mL with deionized water. Store at 2-8°C with a shelf life of 6 months. Discard if the reagent appears cloudy (turbid) or contaminated.
5. Fixation solution (Methanol: Glacial Acetic Acid = 3:1):
   Prepare a ready-to-use fixation solution by thoroughly mixing 30mL of methanol and 10mL of glacial acetic acid.
6. 0.075M KCl Solution:
   Weigh 2.8g of potassium chloride, dissolve in 400mL of deionized water, and complete to 500mL with deionized water. Store at room temperature with a shelf life of 6 months. Discard if the reagent appears cloudy (turbid) or contaminated.
7. Diamidino Phenylindole (DAPI) Counterstain:
   Use commercially available anti-quenching DAPI counterstain.

Instructions

1. Sample Collection and Slide Preparation:
   1. Sample collection: Take 3mL of anticoagulated bone marrow cell samples.
   2. Cell harvesting: Place 3mL of anticoagulated peripheral blood in a 15mL centrifuge tube, centrifuge at 500g for 5 min, carefully discard the supernatant, and resuspend about 500μL of the residue.
   3. Cell washing: Add 5mL of 1×PBS buffer, mix and resuspend the cell pellet, centrifuge at 500g for 5 min, carefully discard the supernatant, and resuspend the cells with about 500μL of the residue; repeat once.
   4. Cells hypotonicity: Add 10mL of hypotonic solution pre-warmed to 37°C and place in a water bath at 37°C for 15-20 min.
   5. Cells pre-fixation: Add 1mL (10% by volume) of fixative solution to the cell suspension after hypotonic treatment. Gently pipette, mix, and centrifuge for 5 min at 500g, discard the supernatant, and resuspend about 500μL of the residue.
   6. Cell fixation: Slowly add 10mL of fixative solution to the cell suspension at room temperature for 10 min, centrifuge at 500g for 5 min, and resuspend the cells with about 500μL of the residue; repeat once (cells may be fixed multiple times until the pellet is washed and cleaned).
   7. Cell suspension preparation: Pipette the supernatant and add an appropriate amount of fixative solution to achieve the correct cell suspension concentration.
   8. Slides preparation: Pipette 3-5μL of cell suspension onto slides and incubate at 56°C for 30 min.
2. Slides Pretreatment:
   1. At room temperature, rinse the glass slides twice with SSC (pH 7.0) solution for 5 min each time.
   2. Place the glass slides in 70% ethanol, then 85% ethanol, then 100% ethanol, and dry for 2 minutes.
3. Denaturation and Hybridization: (Performed in a darkroom)
   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 88°C for 2 minutes (the hybridizer should be preheated to 88°C) and hybridize at 45°C for 2 to 16 hours.

• 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 2min.(Preparation of 0.3% NP40/0.4xSSC: 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.
• 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.
• 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.

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

Manual Approval date & Revision date

V1. 0: Approval date: April 1, 2019.
V1. 1: Revision date: December 7, 2021.