• 00:03 1.
    Paraffin embedded tissue
  • 00:19 1.1
    Deparaffinized
  • 00:18 1.2
    Dehydrate
  • 00:10 1.3
    Air dry
  • 00:32 1.4
    Pre-treatment
  • 00:39 1.5
    Protease treatment
  • 00:28 1.6
    Dehydrate (Room temperature)
  • 00:01 1.7
    Air dry
  • 00:02 2.
    FISH protocol
  • 00:05 2.1
    Mark hybridizing area
  • 00:11 2.2
    Apply 10μl FISH probe for 22mm x 22mm area
  • 00:33 2.3
    Cover with cover glass and seal with rubber cement
  • 00:09 2.4
    Denature
  • 00:18 3.
    Incubation
  • 00:38 4.
    Remove rubber cement Slide into 2X SSC and remove cover glass
  • 00:01 5.
    Counter stain
  • 00:07 5.1
    Apply 10μl DAPI Solution to target area
  • 00:08 5.2
    Put on cover glass Seal with manicure
  • 00:09 6.
    Examine
  • Index
  • Notes
  • Comment
Fluorescence In Situ Hybridization (FISH)
Duration: 05:01, Browse: 1708, Last Updated: 2019-01-09
( http://www.abnova.com ) - FISH is a technique used to identify and localize the presence or absence of specific DNA sequences on cells and tissues. You can use FISH probes for the detection of gene amplification, loss and translocation. Each FISH probe product has a pair of locus-specific, fluorophore-labeled probes originated from a bacterial artificial chromosome (BAC) library.
f674880a25f15868859e253d3d3d1c5c.png
Attachment(s):
Keypoint
  1. 材料準備
  2. 1.
    Preparation of FISH probe
    1. The following FISH probes are ready-to-use, no need of any preparation.
        a. Gene FISH Probe (Cat # FGxxxx)
        b. Split FISH Probe (Cat # FSxxxx)
        c. Translocation FISH Probe (Cat # FTxxxx)
        d. Prenatal FISH Probe (Cat # FMxxxx)
        e. Made to Order FISH Probe (Ca # FAxxxx)
     
     
    2. Chromosome FISH Probe (Cat # FCxxxx) and Subtelomere FISH Probe
        (Cat # FExxxx) are provided in 5x concentrated format, they should be either:
        a. Diluted to 1x with FISH Hybridization Buffer (Cat # U0028 or U0029) before use,
            OR
        b. Mixed with same category of FISH Probes (up to 5 different probes) to use, for example:
    • Combine 2 different probes:
      1 volume of probe 1 (2 uL) + 1 volume of probe 2 (2 uL)
      + 3 volume of FISH Hybridization Buffer (6 uL)
    • Combine 3 different probes:
      1 volume of probe 1 (2 uL) + 1 volume of probe 2 (2 uL)
      + 1 volume of probe 3 (2 uL) + 2 volume of FISH Hybridization Buffer (4 uL)
  3. 2.
    Recommended filter set
    The table below is a recommendation of filter set use:
     
    5c04a2e2ed75a.png
     
    Note: EX. = excitation wavelength; EM. = emission wavelength
  4. 3.
    Protocol selection
    Please follow an appropriate protocol below depend on the sample use, these samples include Paraffin embedded tissue (or FFPE), Frozen tissue and Metaphase spreads.
     
    For Paraffin embedded tissue, we recommended FFPE FISH PreTreatment Kit 1(Catalog #: KA2375 or KA2691 for the pretreatment of Formalin-Fixed Paraffin-Embedded (FFPE) tissue sections.
  5.  
     
    實驗步驟
    Paraffin embedded tissue
  6. 4.
    Deparaffinized
    Xylene 5 min×3 Room temperature
  7. 5.
    Dehydrate
    100% EtOH 5 min×2 Room temperature
  8. 6.
    Air dry
  9. 7.
    Pre-treatment
    Paraffin Pretreatment Solution 95℃ 30 min
    Wash buffer (2×SSC) 5 min×2
  10. 8.
    Protease treatment
    Protease Solution 37℃ 10∼20min
    Wash buffer (2×SSC) 5 min×2
     
    NOTE:
    *Protease Solution Add 500μl protease in 50ml protease buffer
    *Protease preservation One month:4℃ Over one month:-20℃
  11. 9.
    Dehydrate (Room temperature)
    70% EtOH 1 min
    100% EtOH 1 min
  12. 10.
    Air dry
  13.  
     
    FISH protocol
  14. 11.
    Mark hybridizing area
    use diamond pen
  15. 12.
    Apply 10μl FISH probe for 22mm x 22mm area
  16. 13.
    Cover with cover glass and seal with rubber cement
  17. 14.
    Denature
    75℃ 5 min
  18.  
     
    Hybridization
  19. 15.
    Incubation
    Humidified box 37℃ 16 ~ 72 hrs
  20.  
     
    Wash procedure
  21. 16.
    Remove rubber cement Slide into 2X SSC and remove cover glass
    2X SSC Room temp. 5 min
    2X SSC / 0.3% NP-40 73~75℃ 1-2min
    2X SSC Room temp. 1 min
  22.  
     
    Counter stain
  23. 17.
    Apply 10μl DAPI Solution to target area
    *DAPI Paraffin embedded tissue 1500ng/ml
  24. 18.
    Put on cover glass Seal with manicure
  25.  
     
    Examine
    • 00:03 1.
      Paraffin embedded tissue
    • 00:19 1.1
      Deparaffinized
    • 00:18 1.2
      Dehydrate
    • 00:10 1.3
      Air dry
    • 00:32 1.4
      Pre-treatment
    • 00:39 1.5
      Protease treatment
    • 00:28 1.6
      Dehydrate (Room temperature)
    • 00:01 1.7
      Air dry
    • 00:02 2.
      FISH protocol
    • 00:05 2.1
      Mark hybridizing area
    • 00:11 2.2
      Apply 10μl FISH probe for 22mm x 22mm area
    • 00:33 2.3
      Cover with cover glass and seal with rubber cement
    • 00:09 2.4
      Denature
    • 00:18 3.
      Incubation
    • 00:38 4.
      Remove rubber cement Slide into 2X SSC and remove cover glass
    • 00:01 5.
      Counter stain
    • 00:07 5.1
      Apply 10μl DAPI Solution to target area
    • 00:08 5.2
      Put on cover glass Seal with manicure
    • 00:09 6.
      Examine
    Location
    Folder name
    Fluorescence In Situ Hybridization (FISH)
    Author
    賴亮全
    Branch
    賴亮全教授
    Created
    2019-01-09 14:42:59
    Last Updated
    2019-01-09 14:42:59
    Browse
    783
    Duration
    05:01