Immunohistochemistry (IHC)
& Immunocytochemistry (ICC) Support

If you are experiencing difficulties with a Bethyl product, please first consult the Frequently Asked Questions below. If your question isn't answered, please complete the form as accurately as possible. The information is helpful to troubleshoot the differences between the observed results and the expected results. Once submitted, a Technical Support representative will contact you within 1 business day. Please note that all comments and suggestions are taken seriously by Bethyl Laboratories.

IHC, ICC & Multiplex Support FAQs

  • All IHC Antibodies are tested on tissue microarrays.

  • Yes, with immunoperoxidase (DAB) methods you have a permanent slide representing your data. The surrounding tissue is visible to further enhance your data. Also, autofluorescence can confound your data.

  • IF is appropriate for Immunocytochemisty (ICC).

  • Refer to the individual IHC Antibody data sheet. All IHC Antibody data sheets have images demonstrating positive staining.

  • Formalin-fixed, paraffin-embedded tissues should be fixed in formaldehyde no longer than 24 hours. Tissues should not be thicker than 3mm to allow fixative penetration.

  • mIF requires high quality IHC-validated primary antibodies, HRP-conjugated secondary antibodies, tyramide detection fluorophores, and an imaging system capable of separating the fluorophores being used in the assay.

  • Primary antibodies of any host species can be used in mIF, even if the antibodies are from the same host species. It is advisable to begin developing mIF with highly validated IHC antibodies. Bethyl offers and extensive portfolio of IHC validated antibodies. Antibodies from other companies are compatible with Bethyl antibodies but will need to be validated by the researcher.

  • The most common mIF sample is formalin fixed paraffin embedded tissue (FFPE). Since mIF with tyramide detection uses multiple rounds of heat induced epitope retrieval, non-FFPE tissue tends to degrade during the assay.

  • Bethyl antibodies are validated for mIF with the Opal detection system from Akoya Biosciences. Use of other tyramide reagents will require independent validation by the researcher.

  • The mIF protocol outlines the basic optimization parameters. The recommendations given for mIF applications are starting points for assay development. Further optimization of antibodies will be needed using your own tissue. Differences in tissue fixation, epitope retrieval buffer, instruments used for epitope retrieval, and technical skill can impact the mIF assay. The mIF protocol outlines the basic optimization parameters.

  • Order of staining can impact the detection of antigens. Multiple rounds of epitope retrieval can destroy some epitopes. In this case, antibodies recognizing these markers need to be used early in the staining order. Bethyl validated mIF panels will have established an initial staining order. This order should be confirmed on your own tissue before proceeding mIF studies.

  • Fluorophore pairings should be carefully considered when staining for markers that occur in the same cell type, especially if those markers are localized to same subcellular location. In these cases, it is advised to use fluorophores whose spectra are non-overlapping.

  • Bethyl uses steamers for HIER because this method is widely used. Other methods, such as microwave treatment, may be used but will require independent validation by the researcher.

  • The number of targets detectable is dependent on available tryramide fluorphores and the imaging system. Several slide scanners such as the Vectra Polaris offer the capablilty to perform a 9-color scan (8 immunostained markers + DAPI).

    1. Proteins that are highly basic or acidic (charged) may not migrate at their theoretical molecular weight.
    2. Sometimes proteins will retain some secondary structure (re-folding) which can alter mobility.
    3. Post-translational modifications will alter protein mobility.

IHC, ICC & mIF Support Form

Customer Information

Product Information

IHC Method Used

Tissue Sample Information

Immunohistochemistry Method Details

Primary Antibody

Secondary Antibody

Detection Method

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