IHC & ISH

Multiplex IHC 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.

Yes.

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; however, multispectral imaging with a tissue-specific spectral library will resolve this problem.

TRIP has a limited selection of antibodies in its inventory that we will provide and invoice accordingly. If we don’t have an antibody, we will verify the selections made are acceptable for the project’s design so the investigator can place the order. When clients provide the antibody we request the datasheet and storage conditions be provided as well.

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.

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.

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.

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.

Investigators have the option of choosing monoclonal or polyclonal antibodies.  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.

The TRIP Lab performs custom antibody work-up using state of the art automated platform (Roche-Ventana XT).

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.

There are many possible causes of background, including excessive epitope retrieval, endogenous biotin, peroxidase or alkaline phosphatase, antibody dilution factor, and non-specific binding of antibodies.

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.

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 and benefits future researchers.

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 Kari Bonner at kari.bonner@bio-techne.com for any questions about designing your probe with ACD.

We are equipped with RNAscope™ technology from ACD to perform manual 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.

When you are working with Cultured Cells you will be responsible for preparing the cells for hybridization and transporting your slides to TRIP Lab.

These RNAScope Technical Notes will assist you in the steps for preparing your cells.

Cultured Cells Prep Fluorescent

Cultured Cells on Coverslips

Cultured Cells for Chromogenic

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.

Selected tissue should be fixed and processed in a similar manner as your test materials.  TRIP’s ISH work is performed on routine formalin-fixed tissues.

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.

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.

Additionally, TRIP has an Aperio AT2 slide scanner for chromogenic stained slides.  The Aperio can provide whole slide scans up to 40x.

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