ISH: Fluorescence Detection
      

The following protocols are suggested as guidelines for fluorescence 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.

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 at least 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 in between each detection step.

Detect the following labels according to the strategies outlined below. Some options of fluorochrome-labeled avidins are given as examples. The choice of label depends on the individual application. In general, fluorescein Avidin DCS > Texas Red® Avidin DCS > AMCA Avidin D in sensitivity.

To detect biotin-labeled probes:

Incubate with a fluorochrome-labeled avidin (5 µg/ml), such as fluorescein Avidin DCS, Texas Red® Avidin DCS,or AMCA Avidin D. For increased sensitivity, after the first fluorochrome avidin step incubate with Biotinylated Anti-Avidin (5µg/ml), followed by a second incubation with the same fluorochrome-labeled avidin (5 µg/ml).

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

a ) Incubate with Biotinylated Anti-Fluorescein (10 µg/ml), followed by a fluorochrome-labeled avidin (10 µg/ml). This label is usually fluorescein Avidin DCS, but can be Texas Red® Avidin DCS or AMCA Avidin D.

b ) Incubate with Goat Anti-Fluorescein (10 µg/ml), then Fluorescein Anti-Goat (10 µg/ml).

To detect DNP (dinitrophenyl)-labeled probes:

Incubate with Biotinylated Anti-DNP (10 µg/ml), followed by a fluorochrome-labeled avidin (10 µg/ml) such as Fluorescein Avidin DCS, Texas Red® Avidin DCS, or AMCA Avidin D.

To detect Texas Red® or rhodamine-labeled probes:

Incubate with Biotinylated Anti-Rhodamine* (10 µg/ml), followed by a fluorochrome-labeled avidin (10 µg/ml). This fluorochrome is usually Texas Red® Avidin DCS, but can be Fluorescein Avidin DCS or AMCA Avidin D.

Counterstaining and Coverslipping:

Wash slides 2 x 5 minutes in 4x SSC + 0.1% Tween 20 before coverslipping with any one of the following mounting media: VECTASHIELD® Mounting Medium, VECTASHIELD® Mounting Medium with propidium iodide (PI), VECTASHIELD® Mounting Medium with DAPI, VECTASHIELD® HardSet™ Mounting Medium, or VECTASHIELD® HardSet™ Mounting Medium with DAPI.

Multiple Label FISH

In many cases, multiple labels may be used to detect different probes hybridized simultaneously. However, some important points must be taken into consideration.

After hybridization of labeled probes, detect probe separately and sequentially using the procedures outlined above.

Between reagent incubation steps, rinse and block 2 x 3 minutes in the 1x ISH blocking solution.

If the detection procedures for both of the probes include biotin-labeled reagents and (strept)avidin, it is imperative to use (strept)avidin/biotin blocking step after detecting the first probe and before introducing reagents to detect the second probe. This (strept)avidin/biotin block will eliminate any exposed biotin associated with the first probe from being detected by the second set of detection reagents.

In general, choose the most sensitive detection system for the least abundant probe target. This choice may require testing each probe individually with different detection systems prior to using them in multiple label ISH. The order of detecting these probes may also be important in optimizing sensitivity and minimizing color mixing.

For additional information on multiple labeling using enzymatic substrates please see our VECTAGRAM entitled “Labeling of Multiple Antigens”.