Ideal fixation preserves DNA/RNA and tissue morphology at the same time to provide assertive interpretations. Cross-linked fixatives are most commonly used such as paraformaldehyde, formalin and glutaraldehyde.

Fixation needs to be optimized for different target tissues/cells. For example, cryostat sections can be fixed with paraformaldehyde but metaphase chromosome spreads need methanol or acetic acid as a fixative. Cross-linking fixatives tend to mask the target nucleic acid by establishing bridges between the surrounding proteins, therefore, proteinases treatment is necessary to allow better permeabilization of tissue for probe penetration.

• Tissue sections require coated slides in order to secure adherence which are treated with coating solutions (eg. Poly-L-lysine, glutaraldehyde -activated gelatin chrome aluminum);

• Chromosome spreads can be prepared on simple cleaned slides;

• When performing whole-mount ISH (WISH), where whole organisms or organs are used, sterilized and RNase or DNase-free tubes are necessary to avoid desintegration of probe.

Denaturation of probe and target

In in situ hybridization targeted at DNA, denaturation must occur. Denaturation must take into account both signaling intensity and tissue morphology and settle into a compromise. There are two types:

• Alkaline

• Heat - more common due to simplicity and efficiency.

The probe and target DNA may be denatured simultaneously.

A probe is a section of DNA or RNA of variable length (typically 100-1000 bases) which  has to contain a reporter molecule, introduced chemically or enzymatically, that can be detected by affinity cytochemistry. It can be used in DNA or RNA samples to detect the expression of target nucleotide sequences in the probe.

Factors that determine hybridization accuracy:

• Type of probe – oligonucleotides are advantageous due to their small size that enables better tissue penetration, and are single stranded, but their size is also disadvantageous since they cover less target.

• Single stranded vs double stranded probes – single –stranded are more advantageous since probe renaturation cannot occur.

• Probe length – long probes provide maximal hybridization rates but short probes are more feasible for entering cell or tissue matrix.

• Concentration – higher concentration of probe, higher reannealing rate.

   

Pretreatment steps enforce a successful hybridization and decrease occurrence of non-specific staining.

• Proteinases treatment increases tissue permeability to probes.

• Acetylation of sections diminishes non-specific binding of DIG-labeled probes.

• For radioisotopic ISH, adding dithiothreitol (DDT) in the hybridization solution with S-labeled probes reduces background staining significantly.

• Incubation with prehybridization buffer increases hybridization stringency and reduces background staining.

• Innactivation of endogenous enzymes is required for non-isotopic ISH methods since some enzymes, which are used as labels for the probes, are also present in the target tissue/cell. Endogenous Alkaline Phosphatase (AP) can be inhibited with 0.2 N HCl or levamisole; biotin by biotin-blocking agents and, for peroxidase, sections can be treated with 1% H2O2 in methanol.  For more information check external links.

• Using DNase or RNase, depending on the target nucleic acid, to avoid interference during hybridization.

In the hybridization step, denatured DNA reanneals with complementary strands (probes) and this event is associated with their melting point (Tm). Tm represents the temperature at which half he DNA is presented in a single-stranded form. Hybridization depends on:

• pH - the maximum rate of renaturation of DNA is 25ºC.

• Temperature - renaturation is independent between pH values of 5-9. Commonly used buffers contain 20-50mM phosphate and a pH ranging from 6.5 - 7.5.

• Concentration of monovalent cations - monovalent cations such as sodium, interact on the electrostatic repulsion between strands; higher salt concentrations stabilize the hybrid and lower concentrations affect renaturation significantly.

• Presence of organic solvents - DNA denatures at 90ºC - 100ºC in 0.1 - 0.2 M  Na+ which deteriorates tissue morphology in case of microscopic sections. By using an organic solvent (eg.Formamide), DNA melting point is lowered and sections can be hybridized at lower temperatures.

Post-hybridization washes are necessary to eliminate non-specific probe attachment or any residue that can possibly jeopardize clear hybridization results. These are performed with detergents such as 2XSCC. To eliminate non-specific background staining its best to perform high strigency wahes, which have a diluted solution of salt and are higher in temperature. The best results are obtained with high stringency hybridization and similar or lower stringency in washes. Washes are also done between previous stages of in situ protocol to ensure elimination of residues.