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Steps to Better ISH

Geoffrey Rolls, BAppSc, FAIMS

From patient to pathologist, preparing tissue specimens for histological examination requires care, skill and sound procedures. This guide provides practical advice on best practice techniques and simple ways to avoid common errors.

Tips for better ISH are highlighted in this guide. In Situ Hybridization (ISH) is a technique that allows for precise localization of a specific segment of nucleic acid within a histologic section. We hope each step in this guide provides a valuable reminder of good histology practice and helps with troubleshooting when unacceptable results do occur.

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Use High Quality Sections

  Take particular care to use thin, flat sections that have been thoroughly dried onto the slide. Use charged slides for  ISH .

  Uneven, poorly adhering sections stain unevenly with variable background staining.

Image
This poor quality section shows lifting and background staining (HPV).
This poor quality section shows lifting and background staining (HPV).

In Situ Hybridization Ensure Optimal Fixation

  Good quality fixation using known and consistent fixation conditions (fixative type, pH, temperature, time) produces the best results.

  Inconsistent fixation conditions, producing under-fixed or over-fixed tissues, produce variable results and make troubleshooting difficult.

Image
A. shows ISH for kappa light chain mRNA on well-fixed tonsil, this view shows a sharp, strong reaction. B. shows ISH for kappa light chain mRNA on poorly fixed tonsil illustrating a weak reaction.
A. shows ISH for kappa light chain mRNA on well-fixed tonsil, this view shows a sharp, strong reaction. B. shows ISH for kappa light chain mRNA on poorly fixed tonsil illustrating a weak reaction.

Avoid Section Adhesion Problems

  Avoid the use of protein-based section adhesives in the flotation bath (glue, starch, or gelatin), particularly on charged slides.

  Protein-based adhesives can block the surface of the charged slide. This causes inconsistent adhesion and leads to uneven staining due to pooling of  ISH  reagents beneath lifting sections.

Image
A line of thick protein-based section adhesive has stained adjacent to the section (breast,PR).
A line of thick protein-based section adhesive has stained adjacent to the section (breast,PR).

Optimize Wax Removal and Reagent Application

  Take particular care with dewaxing and hydration of sections as well as efficient and uniform distribution of reagents on the specimen surface. This ensures even staining and consistent results.

  Incomplete removal of wax can produce unstained or poorly stained areas in sections. Bubbles retained on the section surface during pretreatment or staining can cause problems.

Image
Bubbles formed during pretreatment at 95 °C have caused uneven staining (HPV).
Bubbles formed during pretreatment at 95 °C have caused uneven staining (HPV).

Choose Probe Carefully

  Choose your probe carefully with regard to its sensitivity and specificity.

  “We buy our probes based on price alone.”

Image
Both sections of tonsil were stained using ISH (BCIP/NBT) for kappa light chain mRNA using oligonucleotide probes from different sources. Section A shows strong staining while section B demonstrates weaker staining with fewer cells stained.
Both sections of tonsil were stained using ISH (BCIP/NBT) for kappa light chain mRNA using oligonucleotide probes from different sources. Section A shows strong staining while section B demonstrates weaker staining with fewer cells stained.

Read Specification Sheets

  Know your probe. Always check the specification sheet to determine the suitability of your method for a particular probe. Carefully control temperature and time to provide optimal hybridization conditions. These must be exactly right to ensure that maximum specific binding occurs.

  No access to the probe data sheets in the laboratory: “We just follow the standard method”.

Image
Both sections of condyloma were stained using ISH for HPV using the same DNA probe but different hybridization conditions. Section A shows strong staining while in section B staining is unsatisfactory.
Both sections of condyloma were stained using ISH for HPV using the same DNA probe but different hybridization conditions. Section A shows strong staining while in section B staining is unsatisfactory.

Optimize Pretreatment Conditions

  Choose appropriate pretreatment and optimization conditions. These will depend on fixation and tissue type.

  Use of the same enzyme pretreatment conditions for different probes may sometimes produce poor results.

Image
This section of colon has been stained with a Poly d(T) positive control probe. The section demonstrates the result of over digestion with Proteinase K. Note the loss of cytoplasmic structure in the mucosal epithelium and heavy background staining.
This section of colon has been stained with a Poly d(T) positive control probe. The section demonstrates the result of over digestion with Proteinase K. Note the loss of cytoplasmic structure in the mucosal epithelium and heavy background staining.

Handle Tissue Carefully

  Careful handling of tissue specimens and prompt fixation will limit the loss of RNA by the action of endogenous RNases.

  Careless handling of tissue specimens and delayed fixation will encourage the loss of RNA by the action of endogenous RNases.

Image
Weak staining due to the breakdown of nuclear RNA by RNases. Tonsil stained with a Poly d(T) positive control probe.
Weak staining due to the breakdown of nuclear RNA by RNases. Tonsil stained with a Poly d(T) positive control probe.

Use Appropriate Detection System

  Choose a sensitive detection and visualization system and optimize incubation conditions.

  A lack of sensitivity in the detection and visualization system can result in very weak or even negative staining even though the probe is bound to a target.

Image
The weak staining in this section of tonsil is due to a lack of sensitivity in the detection system. ISH for lambda light chain using a non-polymer detection kit.
The weak staining in this section of tonsil is due to a lack of sensitivity in the detection system. ISH for lambda light chain using a non-polymer detection kit.

Avoid Reagent Evaporation

  Prevent evaporation of the probe solution and other reagents during incubation. Because of the need for long incubation times drying of the reagents is a common problem. The use of good quality equipment is essential.

  If the probe or other reagents dry out on the section (usually at the edges) it can cause heavy, non-specific staining in areas.

Image
This section of tonsil stained with ISH for kappa light chain has dried out during the formamide post hybridization wash causing inconsistent, non-specific staining.
This section of tonsil stained with ISH for kappa light chain has dried out during the formamide post hybridization wash causing inconsistent, non-specific staining.

Standardize Washing Steps

  Use standardized washing steps throughout (duration, volume and form of agitation). This will ensure consistency of results.

  Results are variable within runs with the same probe and between runs on different days. This can be due to different washing techniques used by different operators.

Image
These sections of tonsil stained with ISH for kappa light chain show how washing affects the result. Section A shows excessive background staining due to poor washing technique while section B was properly washed and has no background staining.
These sections of tonsil stained with ISH for kappa light chain show how washing affects the result. Section A shows excessive background staining due to poor washing technique while section B was properly washed and has no background staining.

Use Appropriate Controls

  Use appropriate controls with every run. This should include known positive tissue and a negative control using a non-specific probe.

  “We only do controls when our method doesn’t seem to work. If we did them for every run people wouldn’t bother to look at them.”

Image
Section of tonsil stained with Poly d(T) positive control probe. The precise staining indicates that the tissue is well fixed and that RNA sequences will be well preserved.
Section of tonsil stained with Poly d(T) positive control probe. The precise staining indicates that the tissue is well fixed and that RNA sequences will be well preserved.

Evaluate Results Carefully

  Know what to look for and where to look when evaluating your test sections and controls after staining. Anyone undertaking ISH should have a fundamental knowledge of the underlying theory of the technique and where to find positive staining.

  If any staining is observed in test sections, it is assumed the stains are satisfactory.

Image
This negative control section of tonsil has gone through all steps of ISH but without the application of a probe. It shows hemosiderin, which has a natural brown color and additionally binds DAB which intensifies the color. This does not represent positive staining.
This negative control section of tonsil has gone through all steps of ISH but without the application of a probe. It shows hemosiderin, which has a natural brown color and additionally binds DAB which intensifies the color. This does not represent positiv

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About the presenter

Geoffrey Rolls, BAppSc, FAIMS

Geoffrey Rolls is a Histology Consultant with decades of experience in the field. He is a former Senior Lecturer in histopathology in the Department of Laboratory Medicine, RMIT University in Melbourne, Australia.

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