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MARTIN BENTZ, STEFAN JOOS AND PETER LICHTER
this is achieved by enzymatic incorporation of modified nucleotides, for
example during a nick translation reaction (Langer et al. 1981).
• Fluorochromes: Directly labeled probes contain fluorochromes as reporter groups (e.g. FITC or rhodamine). Alternatively, labeled probes can be
detected via fluorochromes conjugated to reporter-binding molecules
( e.g. FITC conjugated to avidin or an antibody). Similar to other histochemical detection procedures, different combinations of primary and
secondary reporter-binding molecules can be used for indirect immunofluorescence. lt should be noted that when multiple fluorochromes
are used they must not exhibit highly overlapping emission ranges. Useful combinations of fluorochromes for probe detection and chromosomal counterstain are: rhodamine or texasred with propidium iodide, and
AMCA with DAPI or Hoechst 3325. The use of multicolor hybridizations
is described below.
• Visualization of the probes by epifluorescence microscopy: For many
applications of FISH, a standard epifluorescence microscope equipped
with appropriate filter sets is sufficient. However, data management and
documentation are greatly facilitated by digital imaging microscopy.
This requires specialized equipment, such as a sensitive CCD ( chargecoupled device) camera connected to the standard microscope. Digital
imaging microscopy is mandatory for advanced applications such as recording of weak signals, quantitative measurement of signal intensities
or three dimensional analysis of fluorescently labeled specimens.
Subprotocol 1
Specimen Preparation
Procedure
Formost applications methanol!acetic acid-fixed metaphase preparations
are used. Such metaphase preparations are prepared as described in Chap.
11, then air-dried and treated as follows:
1. Dehydrate in a series of 70%, 90% and 100% ethanol for 5 min at room
temperature.
2. Air-dry slides.
MARTIN BENTZ, STEFAN JOOS AND PETER LICHTER
this is achieved by enzymatic incorporation of modified nucleotides, for
example during a nick translation reaction (Langer et al. 1981).
• Fluorochromes: Directly labeled probes contain fluorochromes as reporter groups (e.g. FITC or rhodamine). Alternatively, labeled probes can be
detected via fluorochromes conjugated to reporter-binding molecules
( e.g. FITC conjugated to avidin or an antibody). Similar to other histochemical detection procedures, different combinations of primary and
secondary reporter-binding molecules can be used for indirect immunofluorescence. lt should be noted that when multiple fluorochromes
are used they must not exhibit highly overlapping emission ranges. Useful combinations of fluorochromes for probe detection and chromosomal counterstain are: rhodamine or texasred with propidium iodide, and
AMCA with DAPI or Hoechst 3325. The use of multicolor hybridizations
is described below.
• Visualization of the probes by epifluorescence microscopy: For many
applications of FISH, a standard epifluorescence microscope equipped
with appropriate filter sets is sufficient. However, data management and
documentation are greatly facilitated by digital imaging microscopy.
This requires specialized equipment, such as a sensitive CCD ( chargecoupled device) camera connected to the standard microscope. Digital
imaging microscopy is mandatory for advanced applications such as recording of weak signals, quantitative measurement of signal intensities
or three dimensional analysis of fluorescently labeled specimens.
Subprotocol 1
Specimen Preparation
Procedure
Formost applications methanol!acetic acid-fixed metaphase preparations
are used. Such metaphase preparations are prepared as described in Chap.
11, then air-dried and treated as follows:
1. Dehydrate in a series of 70%, 90% and 100% ethanol for 5 min at room
temperature.
2. Air-dry slides.