Two major choices need to be made for the preparation of probe: the type of nucleic acid to be used (DNA or RNA, single or double stranded) and the label to be incorporated into the probe. In addition, for certain types of probe, several alternative methods are available for probe synthesis.

A number of different types of nucleic acid probes can be prepared for use in in- situ hybridization:

1. Double stranded DNA probes can be prepared by nick translation, random priming or the polymerase chain reaction (PCR) in the presence of a labeled nucleotide and these probes are denatured before use. Random priming and PCR give the highest specific activities.
2. Single stranded DNA probes can be prepared by primer extension on single stranded templates (3’), by PCR or by the chemical synthesis of oligonucleotides.
3. Oligonucleotides, typically of 20-30 bases in length, can be synthesized and also allow specific probes to be readily designed. These are labeled by incorporating the label during chemical synthesis or by adding a tail of labeled nucleotides. A disadvantage of oligonucleotides is that relatively few labeled nucleotides can be incorporated per molecule of probe and thus they are less sensitive than longer nucleic acid probes.
4. Single stranded RNA probes are synthesized by the use of a purified RNA polymerase (SP6, T7 or T3 RNA polymerase) to transcribe sequence downstream of the appropriate polymerase initiation site. Most commonly, the probe is cloned into plasmid vector so that it is flanked by two different RNA polymerase initiation sites, thus enabling either sense-strand (control) or anti-sense (probe) RNA to be synthesized.

A number of studies have shown that the longer probes give weaker signals, presumably because they penetrate less efficiently into the cross linked tissue. Probes of 50-150 bases give optimal signals for certain tissues. The length of probe can be controlled either during the synthesis reaction or by a subsequent partial cleavage. For nick-translated DNA probes, the length is determined by the amount of DNase in the reaction and for probes labeled by random priming by the concentration of the primer. It is advisable to check whether or not reduction of probe length improves signal for the tissue and preparation method that you are using.

Two major alternatives exist for the label to be incorporated into probe: radioactive labels which are detected by autoradiography, or fluorescent and hapten (non-radioactive) labels which are detected by immunocytochemistry.

Several different isotopes have been used to radiolabel probes for in situ hybridization that differ with regard to the resolution of the signals, the speed of the results and the stability of the probes. 3H-labeled probes offer a sub-cellular resolution of signals, but required long autobiographic exposures. 35S-labeled probes give a resolution of about one cell diameter and give rapid results.

Hapten-labeled probes have a number of assets, including safety, their high stability, rapid results, and a single cell resolution. In addition, it is possible to carryout in situ hybridization in a whole mount. The most sensitive methods involve incorporating a nucleotide derivative with a hapten for which a specific antibody or fluorescent binding protein is available. The tissue is incubated with hapten binding protein coupled to an enzyme, and then the signal is produced by using a substrate for this enzyme that yields an insoluble, colored product. Fluorescent labeling, either of the probe or of the antibody, although less sensitive than the use of enzyme linked antibodies, has been successfully used for chromosomal in situ hybridization. The range of haptens that can be used is at present limited by the commercial availability of antibodies or binding protein. Streptavidin is used to detect biotin-labeled probes and antibodies are available that bind to digoxigenin or fluorescein labeled probes.

1. 10X nick translation buffer [Tris HCl- 0.5 M, MgCl2- 50 mM, Bovine Serum Albumin (BSA)- 0.5 mg/ ml].

2. β-mercaptoethanol (100 mM).

3. dNTP mix (0.5 mM each of dATP, dCTP, dGTP and 0.1 mM of dTTP).

4. Fluorescein 12-dUTP (1 mM).

5. DNase I (10U/ μl ).

6. DNAase dilution buffer (Tris HCl- 0.5 M, MgCl2 - 50 mM, BSA- 0.5 mg/ ml).

7. DNA Polymerase I (10 U/ μl).

8. Salmon sperm DNA (10 mg/ ml).

1. Add 1 μg DNA, 5 μl 10X nick translation buffer, 5μl dNTP mix, 5μl of β mercaptoethanol, 2μl fluorecin 12-dUTP and make final volume 50 μl.

2. Vortex gently, centrifuge and put on ice.

3. Add 1.5 μl DNA Polymerase I (15 U) and 0.45 μl DNAse (stock conc 0.05 U/ μl).

4. Flick the tube.

5. Incubate at 15 ºC for 2 h.

6. Check the size of labeled probe (2 µl) on 2 % agarose gel. Smear intensity should be between 50 to 500 bp.

7. Stop the reaction by heating the tube for 15 min at 70 ºC.

1. Add 1 µl Salmon sperm DNA in 48 µl of labeled DNA.

2. Then add 51 µl of sterile water to make the volume 100 µl.

3. Add 10 µl of 3M sodium acetate solution.

4. Add 250 µl chilled absolute ethanol to the mixture and mix well.

5. Incubate the mixture at -20 ºC for overnight.

6. Centrifuge the mixture at 14,000 rpm for 30 min at 4 ºC.

7. Discard the supernatant, then vacuum dry the DNA for about 30 min.

8. Store the probe in dry form at -20 ºC until use.

1. 10X reaction buffer (10 mM Tris-HCl (pH 8.3), 50 mM KCl).

2. 25 mM of MgCl2.

3. dNTP mix (0.2 mM each of dATP, dCTP, dGTP and 0.13 mM of dTTP).

4. 1.0 mM labeled dUTP.

5. Gene specific primers (10 pmol/ μl).

6. Taq DNA polymerase (1U/ μl).

7. Template DNA (100 ng/ μl).

1. Add 2.5 μl of 10X reaction buffer and 2.0μl of MgCl2.

2. Then add 2.5 μl of each dATP, dCTP, dGTP, 1.63 μl of dTTP and 1.75 μl of labeled dUTP.

3. Add 0.5 μl of each primer 1.0 μl of Taq DNA Polymerase, 0.5 μl of template and make the final volume of 25 μl.

4. Run the program in thermal cycler: initial denaturation at 94 ºC for 5 min; followed by 35 cycles of denaturation at 94 ºC for 1 min, primer annealing at 55 ºC for 1 min, primer extension at 72 ºC for 1 min; with post-cycling extension at 72 ºC for 10 min.

5. Check on 1.5% of agarose gel.

6. Store the probe at -20 ºC until use.

Detection methods are broadly categorized into two major categories:

Direct detection: Direct method applied on the probes that are directly labeled with fluorescent protein linked dUTP or dATP which give fluorescent signal at appropriate excitation range of wavelength.

Indirect detection. Indirect method applies on the probes that are labeled with non fluorescent proteins like biotin or digoxigenin linked dNTP.

This procedure uses successive rounds of Fluorescein Avidin DCS and Biotinylated Anti-Avidin to amplify and detect in situ hybridization signals. The multiple binding capacities of Biotinylated Anti-Avidin provide the potential for significant amplification. This antibody binds to Avidin through the antigen binding sites or through the biotin residues that are covalently attached to the molecule. Following the first application of Fluorescein Avidin DCS, the signal is amplified by incubation with Biotinylated Anti-Avidin, followed by a second incubation with Fluorescein Avidin DCS. This procedure results in the introduction of several more fluorochromes at the target site.

1. After hybridization with biotinylated DNA/ RNA probes, block tissue sections or chromosome spreads for 30 min in 1X ISH Blocking. The effectiveness of the blocking solution may be enhanced by pre-warming the solution to 37 ºC and incubating tissue sections/ chromosome spreads for 30 min or longer at 37 ºC.
   (Note: 5% nonfat dry milk plus 0.1% Tween 20 in 4X SSC used as blocking solution (4X SSC is 0.6 M NaCI, 60 mM sodium citrate, pH 7).
2. Dilute each of the detection reagents, Fluorescein Avidin DCS and Biotinylated Anti-Avidin to 5 µg/ ml in 1X ISH Blocking Solution approximately 30 min before use to minimize non-specific binding.
   (Note: This procedure will require twice the volume of Fluorescein Avidin DCS as Biotinylated Anti-Avidin).
3. Tip off the blocking solution and add the Fluorescein Avidin DCS solution (5 μg/ ml). Incubate for 30 min at room temperature.
4. Wash slide for 2 - 3 min in 1X ISH Blocking Solution.
   If satisfactory sensitivity has been achieved, skip to step 8. For increased sensitivity, continue with steps 5 through 7.
5. Incubate with the Biotinylated Anti-Avidin solution (5 μg/ ml) for 30 min at room temperature.
6. Wash slides for 2 - 3 min in 1X ISH Blocking Solution.
7. It is followed by a second incubation of the same Fluorescein Avidin DCS solution (5 μg/ ml) for 30 min at room temperature.
8. Wash slides in 4X SSC + 0.1% Tween 20 for 2 - 5 min.
9. Apply 40 μl of DAPI II (with antifade solution) to counter stain for 1 h at room temperature and cover with cover slip.
10. Examine under the fluorescent microscope using suitable band filters.

1. Kumar Ravindra, Kushwaha B, Nagpure NS (2012). Fluorescence in situ hybridization (FISH) in fishes: A Practical Approach. NBFGR Publication.