Experiments in primer-mediated in situ labeling techniques

Summary

Primed in situ labeling (PRINS) is a hybridization technique that stains cells and tissues for specific DNA sequences, and it is capable of obtaining the same type of results as FISH.

Operation method

Primer-mediated in situ labeling techniques

Materials and Instruments

Intermediate Chromosome Segregation Phase Anti-Digoxin Antibody
Nucleoside triphosphate Tth or Taq DNA polymerase NaCl EDTA 20 X SSC Tween-20 Ethanol Skimmed milk powder (powder) 10 X dNTP Reaction termination solution Elution solution Closure solution
Coverslips Staining vat Thermostat

Move

I. Standard one-step response

There are two sets of protocols for the PRINS technique, one in which the sample is denatured in hot formamide, as in the FISH technique, and then quenched immediately in a series of gradients of ice-cold ethanol as per 3.4. Alternatively, the sample is denatured by co-heating the specimen and reaction mixture to the denaturing temperature as described below:

1. Determine how large an area of the slide is to be analyzed and select a coverslip and a quantity of reaction mixture appropriate for that area. Use 1ul of reaction mixture per millimeter of coverslip length. Prepare a 6(VL) reaction mixture to completely cover a standard slide and cover with a 24 mmX60 mm coverslip. For smaller areas use smaller amounts of reaction mixture and smaller coverslips.

2. While the reaction mixture is unfolding with the coverslip, place the prepared specimen on a covered thermostat for denaturation at 94°C for 4 min (see Note 4).

3. Transfer the specimen quickly to 55-65°C and incubate for 5-60 min for probe annealing and chain extension. Probe annealing is faster for simple repeating sequences, slower for more complex probes, and chain extension is practically instantaneous, so the annealing time determines the incubation time.

4. Terminate the PRINS reaction by placing the specimen in the preheated reaction termination solution at 55~65°C for 1 min.

5. Transfer the specimen to 50 mL of eluent and elute at room temperature for about 3 min (experiments can be paused at this step, or the specimen can be left in the eluent at 4°C overnight).

6a.If fluorescein-labeled nucleotides were used, the specimen can now be re-stained, blocked, and analyzed by counting under a microscope.

6b.If digoxigenin-labeled nucleotides are used, they need to be colored with an anti-digoxigenin antibody according to 3.6.

II. ddPRINS

Replace the nucleotide species that are not required for DNA synthesis with the correct hybridization probe in the PRINS process with the appropriate dideoxynucleotide (e.g., replace dGTP with ddGTP when synthesizing the human telomere element CCCTAA).

Combinatorial labeling

There are two main different strategies for combinatorial labeling, PRINS reaction first or PRINS reaction second. In PRIN^FISH, PRINS is performed first so that the FISH probe is not involved in the PRINS reaction, and the FISH probe should be added after the specimen has been quenched in ice-cold ethanol. It is important to avoid denaturation of the specimen after PRINS detection, because denaturation of the specimen results in loss of PRINS signal. In PRINS-immunostaining, PRINS should be performed later. After immunostaining the specimen is fixed in 2% paraformaldehyde for 2 min at room temperature and dehydrated with a series of ethanol gradients (70%, 90% and 99%) to enhance the presence of immunostaining prior to PRINS manipulation.

PRINS on denatured specimens

If only formamide denaturation is available, denature the specimen in 70% formamide for 2 min at 70°C if the thermostat is insufficient to provide a heated denaturation temperature, or refer to your laboratory's standard procedure for denaturation by the FISH technique. If PRINS has been performed previously, the specimen may already be denatured. In these cases, it is important to quench the specimen immediately in a series of ethanol gradients at 20°C to keep the target DNA in a denatured structural state.

1. As soon as possible after denaturation, the specimen should be dehydrated in a series of -20°C ethanol gradients (70%, 90%, and 99%) for 3 min each.

2. Remove the specimen from the 99% ethanol and drain off the liquid.

3. Prepare the reaction mixture as described above. If the reaction is a second PRINS reaction, make sure that a differently labeled dNTP is used this time.

4. Annealing and chain extension: Preheat the sample at 55?65°C for lmin. Then add the same preheated reaction mixture to the specimen and spread it over the coverslip.

5. Incubate, terminate and elute as in the previous procedure. At this point the specimen is ready for antibody staining and re-staining/sealing.

6. If a third PRINS reaction is required, repeat steps 1 through 5.

V. Self-primer-mediated in situ labeling (SPRINS)

SPRINS can be performed as in steps 2?5 above (specimens cannot be denatured at any step, so step 1 is not required).

VI. Visualization of Digoxin- or Biotin-Labeled PRINS Products

1. Add 50 μuL of blocking solution containing fluorescein-conjugated anti-digoxin antibody (or streptavidin 2 ng/VL) to the specimen. Cover the specimen with a coverslip and incubate for 30-60 min in the dark.

2. Elute twice with 50 mL of eluent for 5 min each time, at which time the specimen is ready for re-staining and sealing.

VII. Re-staining and sealing

1. Blue repainted DNA can be obtained by using an anti-quencher solution containing 0.4umol/L DAPI.

2. Red replicate DNA can be obtained by using an anti-quencher solution containing PI. 20ul of anti-quencher solution containing 0.5ug/ml is used to block the specimen.

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