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  Section: Biotechnology Methods » Molecular Biology
 
 
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Microslide Preparation of Metaphases for In-Situ Hybridization

 
     
 
Content
Molecular Biology
  The Central Dogma
  Protein Synthesis in Cell Free Systems
  Chromosomes
  Polytene Chromosomes of Dipterans
  Salivary Gland Preparation (Squash Technique)
  Extraction of Chromatin
  Chromatin Electrophoresis
  Extraction and Electrophoresis of Histones
  Karyotype Analysis
  In Situ Hybridization
  Culturing Peripheral Blood Lymphocytes
  Microslide Preparation of Metaphases for In-Situ Hybridization
  Staining Chromosomes (G-Banding)
  Nucleic Acids
  Extraction of DNA from Bovine Spleen
  Purification of DNA
  Characterization of DNA
  DNA-Dische Diphenylamine Determination
  Melting Point Determination
  CsCl-Density Separation of DNA
  Phenol Extraction of rRNA (Rat liver)
  Spectrophotometric Analysis of rRNA
  Determination of Amount of RNA by the Orcinol Method
  Sucrose Density Fractionation
  Nucleotide Composition of RNA
  Isolation of Genomic DNA—DNA Extraction Procedure
  Isolation of Genomic DNA from Bacterial Cells
  Preparation of Genomic DNA from Bacteria
  Extraction of Genomic DNA from Plant Source
  Extraction of DNA from Goat Liver
  Isolation of Cotton Genomic DNA from Leaf Tissue
  Arabidopsis Thaliana DNA Isolation
  Plant DNA Extraction
  Phenol/Chloroform Extraction of DNA
  Ethanol Precipitation of DNA
  Isolation of Mitochondrial DNA
  Isolation of Chloroplast DNA
  DNA Extraction of Rhizobium (CsCl Method)
  Isolation of Plasmids
  RNA Isolation
  Preparation of Vanadyl-Ribonucleoside Complexes that Inhibit Ribonuclease Activity
  RNA Extraction Method for Cotton
  Isolation of RNA from Bacteroids
  Isolation of RNA from Free-Living Rhizobia
  Estimation of DNA purity and Quantification
  Fungal DNA Isolation
  Methylene Blue DNA Staining
  Transformation
  Blotting Techniques—Southern, Northern, Western Blotting
  Preparing the Probe
  Southern Blotting (First Method)
  Southern Blotting (Second Method)
  Western Blotting
  Western Blot Analysis of Epitoped-tagged Proteins using the Chemifluorescent Detection Method for Alkaline Phosphatase-conjugated Antibodies
  Southern Blot
  Southern Analysis of Mouse Toe/Tail DNA
  Northern Blotting
  Restriction Digestion Methods—Restriction Enzyme Digests
  Restriction Digestion of Plasmid, Cosmid, and Phage DNAs
  Manual Method of Restriction Digestion of Human DNA
  Preparation of High-Molecular-Weight Human DNA Restriction Fragments in Agarose Plugs
  Restriction Enzyme Digestion of DNA
  Electroelution of DNA Fragments from Agarose into Dialysis Tubing
  Isolation of Restriction Fragments from Agarose Gels by Collection onto DEAE Cellulose
  Ligation of Insert DNA to Vector DNA
  PCR Methods (Polymerase Chain Reaction)
  Polymerase Chain Reaction
  DNA Amplification by the PCR Method

Purpose
Metaphases are prepared from lymphocytes for in-situ hybridization experiments. The lymphocytes, already uspended in fixative, are dropped from a given height onto cleaned slides. The best preparations of metaphases are obtained when the cells are dropped onto slides immediately after the last wash of the harvesting procedure. The lides are then air-dried, labeled, rinsed in alcohol, and stored in slide boxes at –80°C


Time Required

In 3–4 hours, approximately 80–100 microslides can be prepared for in-situ hybridization.


Materials
  • Microslides, 25 × 75 mm
  • Reagent absolute alcohol
  • Solution A or phosphate-buffered saline
  • Diamond scriber
  • Microslide box, for 100 slides
  • Coplin staining dishes (4)
  • 9" × 9" Technicloth wipes
  • Forceps
Special Equipment
  • Nikon TMS inverted microscope (Frank E. Fryer Company) equipped with 20X and 40X phase contrast objectives and a green filter. The microscope is also equipped with L20 and L40 phase annulus for the LWD condenser.

Procedure
  1. Presoak 6–8 microslides (slides) in absolute alcohol in a coplin staining dish (coplin jar) for several minutes.
  2. Aspirate the old fixative above the cell pellet and resuspend the pellet in 1.5 mL of “fresh” fixative.
  3. Using forceps, remove 1 slide from the coplin jar and wipe dry with a 9" × 9" wipe.
  4. Scratch 2 parallel lines approximately 22-mm apart on the lower third of unfrosted “back” side of slide:


    The scratches will define the area of hybridization of probe DNA and metaphase DNA.

    The room temperature, the barometric pressure, and the humidity will affect the drying time of cells and the quality of the metaphases. It is essential to try to control all the variables and strive to achieve the optimum conditions. The use of vaporizers on days when the air is extremely dry and air conditioners when the room temperature is above 18°C, will help to produce high quality metaphases. Optimum conditions are: room temperature between 15°C–18°C, >50% humidity (a rainy day) and low barometric pressure.
  5. Drop 100 µL of the cell suspension from 3-4" above the slide surface onto the frosted side of the slide. Drop the suspension slowly, 1 drop at a time, moving the pipetman so that no 2 drops fall exactly on the same surface area (but all 100 µL should fall within the scratched area on slide). The fixative will quickly evaporate from under and around the cell and the cell will flatten completely, forcing the chromosomes to spread. Different methods of making slides may speed up or slow down this evaporative process, causing more or less spreading. Throughout the whole process, the cell membrane is present. If a cell breaks open during any stage of the harvest or slide making, some or all of the chromosomes will spill out, causing hypodiploidy with random loss of chromosomes. In severe cases, the slides will have “scattered” chromosomes or completely lack chromosome spreads. To shorten the evaporation time and prevent “scattering” of chromosomes, hold the slide at a 45° angle. Well-spread chromosomes will also have less cytoplasm around each chromosome.
  6. Air-dry the slide either at a slant if the chromosomes are well spread, or horizontal if the chromosomes are not well spread. Quickly scan every slide, using the 20X objective, green filter, and L20 phase annulus. Be careful when viewing the slides; scratching the surface will ruin chromosome material. Check to see if the chromosomes are spread sufficiently and that no cytoplasm is observed (halo or dark area around chromosomes). If cytoplasm is detected, disperse it by washing the cells several more times with “fresh” fixative before preparing more slides. Also, check to see if the suspension is too concentrated or too dilute. Concentrated cell suspensions will produce underspread metaphases, while dilute suspensions are very time-consuming to scan for metaphases.
  7. In a coplin jar, wash 5 or 6 slides at a time in 50 mL of PBS for 5 minutes. Always be careful not to scratch the slides.
  8. After the slides are prepared, wash them in a series of ethanol rinses, 5 minutes each, to rid slides of remaining acetic acid. This is done by increasing the percentage of ethanol in each wash (70, 90, 100%). The chromosomes will harden and become more resistant to conventional banding procedures, e.g., G or Giemsa banding. Therefore, it is important not to wash the slides after slide preparations are done if they will be used for procedures other than in-situ hybridization.
  9. Air-dry the slides vertically for several minutes and transfer them to a microslide box. Wrap parafilm around the box prior to freezing the slides at –80°C (prevents moisture from entering). Slides can be stored for 1 year at –80°C.

    Do not store slides at –20°C or at +4°C, because the quality of the chromosomes
    deteriorates.


 
     
 
 
     




     
 
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