ISH: Enzyme-Based Detection
      

The following protocols are suggested as guidelines for enzyme-based detection of in situ hybridization probes.

Pre-Blocking (Post-Hybridization):

Block tissue sections or chromosome spreads for ≥ 30 minutes in 1x in situ blocking solution (5x ISH Blocking Solution, Cat. No. MB-1220). 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 minutes or longer at 37 ºC.solution to 37 ºC and incubating tissue sections/chromosome spreads for 30 minutes or longer at 37 ºC.

Note: 5% nonfat dry milk plus 0.1% Tween 20 in 4x SSC can be used as an alternative blocking solution. (4x SSC is 0.6 M NaCl, 60 mM sodium citrate, pH 7.0.) However, non-fat dry milk can contain variable amounts of biotin which could reduce staining if used as a diluent for (strept)avidin conjugates.

The concentrations of the Vector® reagents listed below are recommended for 30 minute room temperature incubations unless otherwise indicated. Each protocol should be optimized for the individual application.

Detection reagents can be diluted in 1x ISH blocking solution for approximately 30 minutes before use to further reduce any non-specific binding.

Slides can be washed for 2 x 3 minutes in 1x ISH blocking solution between each detection step.

The most sensitive peroxidase and alkaline phosphatase substrates are described here although alternative substrates may be used. Before applying the substrate solution, wash slides 2 x 5 minutes in TBS (Tris buffered saline: 0.1 M Tris, 0.15 M NaCl, pH 7.5). Enzyme substrate solutions should be made according to the kit instructions.

To detect fluorescein-labeled probes, select from the following three options:

a) Incubate with Alkaline Phosphatase Anti-Fluorescein (5 µg/ml), then BCIP/NBT substrate. (For overnight incubation with BCIP/NBT, use Alkaline Phosphatase Anti-Fluorescein at 0.2 - 2.0 µg/ml.)

b) Incubate with Biotinylated Anti-Fluorescein (5 - 10 µg/ml), then Alkaline Phosphatase Streptavidin (1 - 5 µg/ml),followed by BCIP/NBT substrate. (For overnight incubation with BCIP/NBT, use Biotinylated Anti-Fluorescein at 0.2 - 2.0 µg/ml and Alkaline Phosphatase Streptavidin at 0.3 - 0.5 µg/ml.)

c) Incubate with Biotinylated Anti-Fluorescein (5 - 10 µg/ml), then VECTASTAIN® Elite ABC Kit (Standard), followed by TMB substrate.

To detect biotin-labeled probes, select from the following three options:

a) Incubate with Alkaline Phosphatase Streptavidin (1 - 5 µg/ml), then BCIP/NBT substrate. (For overnight incubation with BCIP/NBT, use Alkaline Phosphatase Streptavidin at 0.3 - 0.5 µg/ml.)

b) Incubate with Alkaline Phosphatase Anti-Biotin (5 - 10 µg/ml), then BCIP/NBT substrate.

c) Incubate with VECTASTAIN® Elite ABC Reagent (Standard Kit), then TMB substrate.

To detect DNP (dinitrophenyl)-labeled probes, select from the following three options:

a) Incubate with Alkaline Phosphatase Anti-DNP (5 µg/ml), then BCIP/NBT substrate. (For overnight incubation with BCIP/NBT, use Alkaline Phosphatase Anti-DNP at 0.2 - 2.0 µg/ml.)

b) Incubate with Biotinylated Anti-DNP (5 - 10 µg/ml), then Alkaline Phosphatase Streptavidin (1 - 5 µg/ml), followed by BCIP/NBT substrate. (For overnight incubation with BCIP/NBT, use Biotinylated Anti-DNP at 0.2 - 2.0 µg/ml and Alkaline Phosphatase Streptavidin at 0.3 - 0.5 µg/ml.)

c) Incubate with Biotinylated Anti-DNP (5 - 10 µg/ml), then the VECTASTAIN® Elite ABC Reagent (Standard Kit), followed by TMB substrate.

To detect Texas Red® or rhodamine-labeled probes, select from the following three options:

a) Incubate with Alkaline Phosphatase Anti-Rhodamine* (5 µg/ml), followed by BCIP/NBT substrate. (For overnight incubation with BCIP/NBT, use Alkaline Phosphatase Anti-Rhodamine at 0.2 - 2.0 µg/ml.)

b) Incubate with Biotinylated Anti-Rhodamine* (5 - 10 µg/ml), then Alkaline Phosphatase Streptavidin (1 - 5 µg/ml), followed by BCIP/NBT substrate. (For overnight incubation with BCIP/NBT, use Biotinylated Anti-Rhodamine at 0.2 - 2.0 µg/ml and Alkaline Phosphatase Streptavidin at 0.3 - 0.5 µg/ml.)

c) Incubate with Biotinylated Anti-Rhodamine* (5 - 10 µg/ml), then the VECTASTAIN Elite ABC Kit(Standard), followed by TMB substrate.

* Alkaline Phosphatase Anti-Rhodamine (MB-1920) and Biotinylated Anti-Rhodamine (BA-0605) bind most rhodamines, including Texas Red®

Additional Enzyme Substrate Information

Alkaline Phosphatase Substrates:

BCIP/NBT (blue-violet) is one of the most sensitive enzymatic substrates because of the significant increase in reaction product with longer incubation times (e.g. overnight). Adding 10% polyvinyl alcohol (31 - 50,000 MW) to the substrate solution may produce a more discrete reaction product. BCIP/NBT can be dehydrated and cleared, but is not compatible with all permanent mounting media. VectaMount™ mounting medium is well suited for BCIP/NBT permanent mounting because it minimizes crystal formation in the mounted sections.

Vector® Red produces an intense red reaction product and, unlike other red alkaline phosphatase substrates such as Fast Red, Vector® Red can be permanently mounted. The Vector® Red reaction product is also highly fluorescent and non-fading. Adding 0.4 M NaCl to the substrate solution may increase sensitivity.

Peroxidase Substrates:

Vector® NovaRED™ (red), DAB (brown), and Vector® VIP (purple) are sensitive peroxidase substrates that provide discrete reaction products and are also visible using darkfield or electron microscopy. TMB (blue), the most sensitive peroxidase substrate, provides a more diffuse reaction product than the other peroxidase substrates. TMB reaction product should not be washed for longer than 10 minutes (including counterstaining and rinsing) to avoid a decrease in sensitivity.

Multiple label In Situ Hybridization (ISH) or Immunohistochemistry /In Situ Hybridization (IHC/ISH):

DAB and Vector® Red are recommended for sequential, multiple label ISH or IHC/ISH, because of their stability throughout in situ hybridization procedures.

Counterstaining and Coverslipping

Wash slides according to Vector Substrate Kit instructions before counterstaining, dehydrating, clearing, and coverslipping. BCIP/NBT and TMB substrates are compatible with Vector® Nuclear Fast Red counterstain. Vector® NovaRED™,DAB, and Vector® Red are compatible with Vector® Hematoxylin and Vector® Hematoxylin QS. Vector® NovaRED™‡, Vector® VIP, DAB, BCIP/NBT, and Vector® Red are compatible with Vector® Methyl Green Counterstain. ‡A slight color change in Vector® NovaRED™ reaction product may be seen when using Methyl Green.