IHC & ISH

TRIP offers an array of assays either for your immunohistochemistry (IHC) or in situ hybridization (ISH) experimental needs. IHC will allow you to look into the spatial localization of proteins in tissue while ISH allows you to study different types of RNA localization. We offer both chromogenic or fluorescence (ISH or FISH) modalities for all techniques. Currently, we can stain up to 4 colors (chromogenic IHC) or 7 colors (IF) including counterstain. For ISH, we can do up to 3 colors and for FISH up to 4 colors, including counterstain. All ISH/FISH fees vary per project. Please refer to our TRIP Fee Schedule for pricing. These services need a project design meeting before commencing work. Please submit an iLab Project Design Inquiry Request.

If you are interested in multiplexing and using the Vectra or Nuance microscope systems for multispectral imaging, please contact us at trip@pathology.wisc.edu with any questions or to schedule training.

RESOURCES

Antibodies TRIP Has Experience With (Updated Annually)

IHC-Tissue Imaging by Wei Huang 2016

ISH by Ricardo Lloyd 2016

ISH FAQs

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How can I design an ISH probe?

There are several companies that offer ISH probes. We have partnered with Advanced Cell Diagnostics (ACD), so they can assist you in designing the right probe for your experiment. Please contact Stephanie Craig at stephanie.craig@bio-techne.com or Kari Bonner at kari.bonner@bio-techne.com for any questions about designing your probe with ACD.

What type of ISH assay do you perform at TRIP?

We are equipped with RNAscope™ technology from ACD and Roche-Ventana to carry out either manual or automated ISH/FISH assays. RNAscope™ is a novel, multiplex, nucleic acid, in situ hybridization technology. This technology has overcome the pitfalls of the conventional ISH/FISH-based in situ RNA detection techniques, such as a lack of robustness and sensitivity to reliably detect the expression of most human genes. The assay consists of a set of target probes and a signal amplification system composed of preamplifiers (PreAMP), amplifiers (AMP), and label probes.

Furthermore, multiple distinct amplification systems have been built that do not cross-react with each other and recognize unique sequences on different sets of target probes allowing for the simultaneous detection of multiple RNA targets.

What is the estimate cost for a 1-color ISH experiment?

The ISH kits and probes are to be provided by the costumer since they are project dependent. ISH supplies for 20 samples plus 1 custom probe is estimated to cost about $2000. If the probe is already available in the ACD catalog, then it will be less.

Does TRIP provide control tissue for optimization, or do I need to provide it?

TRIP has a wide selection of human tissue for optimization however, we have limited mouse tissue. If we do not have the appropriate tissue we will request that tissue be provided, whether in block or section format.

How do you select control tissue?

Tissue selection for optimization may overlap with tissue used as a staining control. For optimization purposes you want to see not only strong staining but also weak staining of target tissue, which is why it is good practice to stain at least 2-3 different tissues (or different cases of the same type of tissue) to verify a range of staining intensity.

Select tissue that has been fixed and processed in a similar manner as your test materials.  TRIP’s ISH work is performed on routine formalin-fixed tissues.

If optimization doesn’t go well at first, test on 1-2 additional tissues. This allows ruling out the initial tissue selection as the problem before choosing a different probe.

Running positive or negative controls with test slides is up to the client’s discretion and selection. Many clients count on internal controls within the test tissue.

How can I image my stained slides?

TRIP Lab has two microscopes with multispectral imaging capabilities.  The Nuance and Vectra systems will produce high quality images. These images can be unmixed with Nuance software resulting in images with no/little background emphasizing your true ISH signal. Go to our Imaging section for more information.

How can I analyze my ISH data?

Currently, TRIP offers InForm software for you to quantify the amount of signal present throughout the images. There is also open source software, such as ImageJ/FIJI plugins that can help you. Below are some guides provided by ACD on how to do image quantitation.

Bright Field Quantitation with ImageJ

Fluorescent Quantitation RNAscope

Can you rush my project if I have a short timeline?

IHC-IF can be rushed on any commercially available or home-brew antibodies, which will incur a 20% increase in the billing of the project.

  • Set up fee includes an initial consultative meeting, administrative and experimental setup costs.
  • Consultation includes assistance in finding antibodies, appropriate controls, etc.
  • Includes cost of all non-antibody reagents, technician time and consumables.
  • Investigator must supply the antibody or antibody cost (if provided/purchased by TRIP), slides or blocks for staining.

What is the workflow for antibody customization?

The TRIP performs custom antibody work-up using state of the art automated platform (Roche-Ventana ultra). Related fees include a one-time set-up fee per antibody (covers: Initial consultation, experiment setup and administrative costs) plus a charge per slide for the optimization run(s) conducted during antibody work-up and eventual staining of the actual slides in question. The investigator supplies the antibodies or pays the cost if TRIP orders it. TRIP does carry a number of clinical/research grade in-house commercially available antibodies and charges a flat fee of $5 per slide. Each case is unique and fees may change depending on the scope of the request or subsidies. Quotes can be provided on request.

A typical antibody workflow may look like this:

  1. Consult with investigator
  2. Obtain candidate antibody and source positive and negative control tissues or cut sections
  3. Perform initial staining run on controls
  4. Use information from initial run to conduct additional optimization/validation run(s)
  5. Once antibody has been validated and optimal conditions are determined for staining, run pilot staining runs on the investigator’s tissue of interest

There are no guaranteed results from using this service. Fees will be accessed on work done and not outcome. Please provide primary antibody spec sheets with requisition. For best results please have your tissue processed and sectioned using our TMA/Histology service.

Immunostaining is notoriously prone to generating false negative and false positive results. Unless there are well validated antibodies available for the target of interest, we STRONGLY recommend that the investigators opt for TRIP’s robust positive and negative controls to be used during the initial antibody work-ups.

Furthermore, if there are no known well validated antibodies for your target, we advise trying multiple different antibodies during the work-up phase in order to increase the probability of finding one that performs well.

What are examples of negative and positive controls for antibody validation?

Examples of tissue controls for antibody validation:

  • In-House Tissues that are well documented to endogenously express no/low versus high amounts of the target
  • Diseased versus normal tissues to appreciate differences in expression of the target

Examples of reagent controls for antibody validation:

  • IgG or corresponding immunoglobulin serum antibody that matches primary antibody of interest as a negative control
  • Omission of primary antibody and adding secondary antibody only as a negative control

Without such rigorous controls, it can be very difficult to interpret any staining eventually generated on your actual tissues of interest.

FAQS concerning IHC and TRIP IHC Services

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Why do I need an IHC project design meeting to initiate a project?

The informational meeting is an opportunity for clients and TRIP personnel to go over the iLab form to verify details and agree on the best path to reach your goal. This also allows clients an opportunity to ask questions and for staff to share their knowledge and experience.

Do I need to submit cut sections, or will TRIP section blocks for IHC?

Test tissue can be submitted either way.

If blocks containing bone are being submitted, indicate if the tissue was decalcified prior to processing and embedding.

If cut sections are being submitted, be sure the sections have been cut at a consistent thickness, picked up on charged slides and not previously “baked” to remove paraffin. If slides have been previously cut it is best if they have been either stored in the refrigerator or freezer as there are publications addressing the loss of antigenic reactivity in stored paraffin tissue sections.

What is multiplex IHC (mIHC)?

mIHC is an efficient and cost effective way to expand the amount of information you can achieve from immunostaining while conserving tissue.  It allows observation of the localization of markers, including co-localization. Both IHC and ISH can be run on the same tissue, allowing for information on both protein and DNA/mRNA targets.

What factors should be considered when planning a mIHC project?

Optimal selection of antibodies would be based on each being from a different host species. Since this is not always possible with current commercial sources and time restraints, there are ways to work around this by carrying out elution (stripping)/denaturing steps between antibodies of the same species to prevent cross-reactivity.

When selecting of detection systems it is important to take into account how the color combinations will look together, both visually and spectrally, and whether the detection of a less abundant marker might be hindered by a more dominant marker, especially if co-localized.

Can antibodies from the same species be used in mIHC?

While not optimal, it is possible with the appropriate validation and treatment steps to elute (also called “strip”) residual primary antibody and detection reagents through heat or acid treatments or a combination of both. It should be kept in mind that taking some tissues through multiple rounds of denaturing can affect the tissue integrity.

How do I decide between fluorescent and chromogenic IHC?

Since both have advantages and disadvantages, the choice needs to be made on the basis of the project goal. In general, chromogenic IHC might be considered more sensitive (especially when paired with amplification steps) but when looking for co-localization of two or more target proteins in the same cell compartment fluorescent markers have the edge.

Chromogenic:

  • Chromogenic detection does not need a specialized microscope or filters.
  • Permanently mounted slides exhibit superior stain stability and are easily stored at room temperature. This stability allows the user to revisit the slides without fear of diminishing signal and subsequent loss of information.
  • Chromogenic detection can be problematic when looking for co-localized markers unless using spectral imaging to separate the colors.
  • The intensity of chromogen deposition at the individual cell level cannot be assumed to be quantitative.

Fluorescence:

  • When imaged, quantitation with fluorescence is more accurate due to clear, linear signal localization. There is no blend or overlap of signal to consider.
  • Fluorescence has a greater color selection and narrower emission spectra than chromogenic methods.
  • Fluorescence requires a microscope with appropriate light source and filters to view results.
  • Stain quality is subject to temperature and photo-bleaching of fluorophores.
  • Morphology is not easily appreciated in comparison to IHC-chromogenic.
  • Autofluorescence can interfere with interpretation at the visual level- multispectral imaging will resolve this problem.

Does TRIP provide the primary antibodies I need or do I need to buy them?

If TRIP purchases the antibodies or provides aliquots from its inventory we will verify the selections are acceptable to the investigator during the project design phase and invoice accordingly. If the client is providing the antibody we request that the datasheet and storage information be provided. Either method is acceptable.

Does TRIP provide control tissue for optimization, or do I need to provide it?

TRIP has a wide selection of human tissue for optimization however, we have limited mouse tissue. If we do not have the appropriate tissue we will request that tissue be provided, whether in block or section format.

How do you select control tissue?

Tissue selection for optimization may overlap with tissue used as a staining control. For optimization purposes you want to see not only strong staining but also weak staining of target tissue, which is why it is good practice to stain at least 2-3 different tissues (or different cases of the same type of tissue) to verify a range of staining intensity.

Select tissue that has been fixed and processed in a similar manner as your test materials.  The majority of TRIP’s IHC work is on routine formalin-fixed tissues.

If optimization doesn’t go well at first, test on 1-2 additional tissues. This allows ruling out the initial tissue selection as the problem before choosing a different antibody.

Running positive or negative controls with test slides is up to the client’s discretion and selection. Many clients count on internal controls within the test tissue.

How do I select primary antibodies for my project?

Antibody selection can be discussed in the project design meeting if a decision has not already been solidified based on project goals and prior literature searches. There are markers that are well reported in reputable publications that make excellent choices, while some antibodies may have reported history but not in the same tissue or disease state as your project goals, while others have minimal to no references to base your decision on.

While investigators have the option of choosing monoclonal or polyclonal antibodies, sometimes they won’t have a choice.

Monoclonal antibodies have greater epitope specificity and lower true background, but are often times less sensitive and require more rigorous pretreatment to unmask epitope sites.

Polyclonal antibodies have higher sensitivity through recognition of multiple epitopes, but have disadvantages such as more batch-to-batch variability and higher background.

TRIP can provide clients with information on preferred vendors and clones from our previous successful results.

What is signal amplification?

There are instances in IHC where a marker is either scarce in the target tissue or is a low-affinity antibody. In these instances, in order to “see” the antibody-linked antigenic site more signal deposition is required for detection. TRIP has a variety of options to “amplify” the signal for better visibility.

Why does TRIP use multiple types of diluents?

Traditionally, the routine buffers used for diluting antibodies have been either a Tris or PBS-based buffer, both containing BSA.  Research has shown that there are some antibodies that don’t respond well when diluted in these buffers. As a result, we work with several diluents with different pHs, buffer concentrations and ionic strengths, selecting the best match for each antibody.

Why does TRIP provide both automated and manual IHC?

The majority of TRIP’s IHC volume is automated, providing greater efficiency and standardization. There are a few long-term projects that are done manually for continuity.  While there have been instances where an antibody responds better to some of the manual pretreatment options, the decision to automate rests with TRIP.

What causes background staining?

There are many possible causes of background, including excessive epitope retrieval, endogenous biotin, peroxidase or alkaline phosphatase, improper antibody dilution factor, tissue drying during staining process, old slides, and non-specific binding of antibodies. Each possible source should be considered.

TRIP Lab uses commercially-labeled polymer systems which rarely display background staining. Often times the perceived background can be diffused antigen in the tissue section, or real staining that is directly tied to the selected antibody.

What causes tissues to fall off or fold during IHC protocols?

There are several causes attributed to tissue falling or folding:

  • The antigen retrieval method and/or buffer is too harsh for the target tissue.
  • Citrate buffer is usually easier on tissue than the high pH buffers but might require longer time or higher temperature to achieve maximum epitope unmasking.
  • If using an enzyme, either reduce time or select a milder protease.
  • Poorly fixed and processed tissue.
  • Tissue sectioning problems-i.e.: sectioning with a dull blade.
  • Age of slides and/or cut sections. Charged slides do deteriorate over time, so it is best to not order more than what you will use in the next 6 months to a year.
  • Some tissues are more prone to fall off, especially breast, necrotic tissue, frozen sections, bone and tiny specimens.

What happens to residual antibody and tissue sections or blocks at the end of a project?

All materials will be returned to the client at the end of the project. Any antibody the client wishes to donate to the TRIP inventory is greatly appreciated.