In Situ Hybridization Support—Troubleshooting

Here is a general CISH and Rembrandt CISH kit troubleshooting checklist:

1) Check all items in the Rembrandt kit in order assure that the problems are not caused by assembly mistakes.

2) Check if the probes match the conjugates, that is, biotin-labeled probes should only be used with an anti-biotin conjugate, while digoxigenin-labeled probes should only be used with an anti-digoxigenin conjugate.

3) Ensure that the conjugate matches the enzyme substrate, that is, HRP should be used with DAB and AEC, while alkaline phosphatase goes with NBT/BCIP and Fast Red.

4) Ensure that the correct wash solutions are being used. For example, in the Rembrandt CISH kits, the wash solutions (called PanWash) used for HPV probes and for CMV and EBV probes are different.

5) Confirm that the enzyme conjugate being used is active. This is easily done by mixing 1 drop of conjugate with 1 drop of substrate in a small reaction tube. You should notice a definite color change in the tube within a few minutes. If you see a positive reaction, you will then know that the conjugate is still active and that the substrate reagent is behaving as expected.

6) Check all incubation temperatures with a validated thermometer. You can check the temperature of the hot plate by putting an empty glass slide on the hot plate and then placing the bulb of the thermometer on top of the slide. Move the slide to different areas of the hot plate because temperatures of the may vary from place to place on the surface. 

If there have been no issues related to items 1-6 above, then you should verify that that there were no mistakes in the actual CISH protocol. The most important steps in the procedure are the heat treatment step for antigen retrieval, the pepsin digestion step, the denaturation step, the hybridization step, the detection step, and the mounting step. 

1) For the heat treatment (heat-induced epitope retrieval) step, it is recommended to heat the sections for 15 minutes starting from the time the pretreatment buffer reaches 98°C. 

2) For the pepsin digestion step, between 3-10 minutes at 37°C is recommended for most tissues. Make sure to prevent evaporation during this step. However, the enzyme pretreatment conditions may need to be adjusted according to the properties of the sample tissue. For example, we have observed that over-digestion can weaken or eliminate the CISH signal and prevent counterstaining of cell nuclei. Under-digestion may also decrease of eliminate the CISH signal, but you will still be able to counterstain the nuclei.

3) The denaturation step should be done at 95 ± 5°C for 5-10 minutes. During denaturation, the sections should be cover-slipped. We have found that the denaturation step is best performed on a hot plate on which no more than 5 slides are incubated simultaneously. They should be covered with a lid of some kind in presence of a small amount of pre-warmed water, which provides a moist environment. 

4) The hybridization step should be conducted at 37°C and the slides should be cover-slipped after the probe is applied. For best results, the optimum incubation time is 16 hours (overnight). Run the hybridization in a closed chamber in presence of a small amount of pre-warmed water, which provides a humidified environment. Make sure to mix the probe well before applying it to the slides. After hybridization, remove the coverslips by soaking the slides in PBST buffer. Then wash the slides in PBST and incubate them with the correct pre-warmed Rembrandt TBS wash buffer at 37 ±2°C for 15 minutes. The slides are then rinsed in TBS wash buffer prior to the stringent wash step. For RISH only, warm up the probe to 85°C for 5 min before using it.

5) Make sure that the stringent wash is performed properly. After hybridization, rinse the slide briefly at room temperature with SSC buffer, and then immerse them for 5 minutes in SSC at 75°C. Increase the temperature by 1°C per slide for more than 2 slides, but do not exceed 80°C. After the stringent wash, rinse the slides with TBST. Washing with any other reagents such as PBS without Tween 20 or distilled water can lead to unwanted background. 

6) For staining, incubate the slides with the enzyme conjugate at 37°C for 30 minutes and then rinse with PBS buffer 3 times for 1 minute each. Then, rinse the slides in distilled water and incubate with the substrate solution at 37°C for 5-15 minutes. We recommend that you check the intensity of the staining of your positive control sample under the microscope signal at 2 minute intervals. The moment you start to see background staining appear is the moment to stop the reaction. This is done by rinsing the slides in distilled water. You can then apply a light counterstain, if desired.

7) Remember that dark counterstaining with hematoxylin can mask a positive signal with DAB and NBT/BCIP, in particular. We suggest a counterstaining time with hematoxylin from 5 seconds-1 minute. We recommend Mayer’s hematoxylin (Cat. No. 00-8011) for the counterstaining step. 

8) During all of the steps in the CISH protocol, make sure that the tissue sections do not dry out at any time.

Background staining in CISH can be caused by the nucleic acid probes used. If your probes contain a lot of repetitive sequences like Alu or LINE elements, this can elevate background staining. If necessary, you will have to block probe binding to these repetitive sequences by adding, for example, COT-1 DNA during the specific CISH hybridization. During the various protocol steps that specify using PBST, washing the slides with distilled water or PBS without the Tween™ 20 can lead to elevated background. High background staining can occur if the stringent wash step was inadequate. Make sure that the temperature of the SSC stringent washing solution is 75-80°C. 

Finally, we would recommend following the staining reaction microscopically instead of by eye, as this can lead to background staining that is higher than necessary. The moment that background staining appears is when the reaction should be stopped by rinsing the slides in distilled water. Apply a light counterstain if required.

Many factors can influence the performance of the ISH procedure: 

Low or no staining intensity can be due to improper tissue sample handling. For example, a long time interval between obtaining the tissue and fixing it can degrade the CISH, FISH (and IHC) results. If a tissue specimen is too large for the amount of fixative used, or the fixation time is too short, CISH and FISH signals and tissue morphology can both suffer. 

Similarly, the quality of CISH or FISH results will reflect if the tissue sections were too thin or too thick or they did not adhere well to the slides because the incorrect tissue adhesive was used. Like any IHC technique, CISH/FISH staining intensity will be affected by the efficacy of the de-paraffinization step, by the effectiveness of the antigen retrieval step, by the integrity of the detection reagents, and by the incubation times and temperatures used. Also, the validity of your results depends on being able to compare the results of your samples to those obtained with the appropriate controls. The best way to assure that your results are valid is to always run positive and negative control slides whenever you perform CISH or FISH. 

The outcome of a CISH and FISH procedure is also affected by the sensitivity of the method and the amounts of DNA or RNA target in the sample. For example, if the target DNA or RNA content in the tissue sections is too low, a false negative reaction may be obtained. RNA FISH probes are the best option for hybridization to mRNA, rRNA, or other RNA targets. DNA FISH probes can also be used to detect RNA targets, but they do not hybridize as well to mRNA and other RNA target molecules. DNA FISH probes are routinely used to detect gene loci on chromosomes. RNA FISH probes can also be used to detect genes on chromosomes, but DNA FISH probes are more robust for this application.

In FISH, the probes are generally from 300 to 1500 bp in length. To adequately image a gene or other target, the FISH probes must cover a contiguous length of ˜10 kbp. Shorter strand lengths or low-abundance DNA or RNA targets may be detected with the use of fluorescent amplification methods, such as tyramide signal amplification (TSA). 

NBT/BCIP (along with Fast Red) is a substrate for alkaline phosphatase, not HRP. The precipitating substrates for HRP are DAB and AEC.

Insufficient stringency washing can lead high DAB background staining on the slides. We recommend using a 1X SSC buffer at a temperature of between 75-80°C for the wash step. It is also a good idea to increase the incubation temperature by 1°C per slide if you are staining ≥2 slides. However, we recommend that you do not exceed 80°C. Stringent washes performed at temperatures higher than this can decrease or even eliminate the CISH signal. 

During the actual IHC staining portion of the CISH protocol, wash the slides with 1X PBS containing 0.025% (v/v) Tween™ 20 (PBST). If desired, the progress of the DAB staining step can be checked under the microscope using the 20X objective. It is typical for a clearly visible signal to appear after about 20 minutes of incubation.

We do not recommend using a dark counterstain with CISH detection. For example, the blue color from typical hematoxylin counterstaining can mask the brown DAB product and the dark blue NBT/BCIP CISH signals. Counterstaining in Mayer’s hematoxylin (Cat. No. 008011) for 5 seconds to 1 minute is usually adequate for enhancing contrast and improving specimen visibility.

Finally, when you mount your sections prior to imaging, we recommend using Histomount (Cat. No. 00-8030) as the mounting medium. When the Histomount is applied, your sections should still be wet, but it is important to avoid bubbles when you are applying the coverslips.