Start Your Project

🛍️Explore our Promotions page to discover current deals, limited-time offers, and more!🌟

Immunohistochemistry Troubleshooting

ProSci Inc has answers for all your Immunohistochemistry(IHC) troubleshooting needs. Consult our Immunohistochemistry protocol and these immunohistochemistry tips for all stages of IHC protocol.

High Background or Non-Specific IHC Staining

For IHC troubleshooting high background or non-specific IHC staining consider the following:

Endogenous Enzyme

Block endogenous enzyme (peroxidase) activities using 3% hydrogen peroxide prior to incubation of primary antibody if HRP-based detection system is being used.

Blocking

Increase the blocking incubation duration and consult Prosci technical support for the recommended blocking reagent.

Antibody Dilution/Diluent

Run a serial dilution test to determine the optimal dilution. Consult Prosci product datasheet for the recommended dilution. Higher dilution of primary antibodies can reduce the non-specific binding.

Secondary Antibody Cross-Reactivity

Run a secondary control without a primary antibody. If positive, change your secondary or use a secondary antibody that has been pre-absorbed against the Ig of the species of your samples. Alternatively, reduce the concentration of the biotinylated secondary antibody.

Primary Antibody (Mouse on Mouse tissue)

Use a primary antibody raised against a different species than your tissue. Or use a biotinylated primary antibody and a conjugated streptavidin for the detection system.

Amplification/Detection System

Too much amplification or substrate can lead to non-specific staining or high background. Reduce amplification/substrate incubation time and dilute the secondary antibody or amplification kit.

Washing

Wash extensively in PBS between all steps.

Incubation Temperature

Incubate slides overnight at 4°C.

Slides dried out

Keep slides at high humidity and avoid sections being dried out.

Conjugating the Peptide to Enhance Immunogenicity

The two most common conjugates, also known as carrier proteins to which antigens attach, are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). These two molecules are highly immunogenic, meaning they are good at activating humoral response that creates antibodies directed against the antigen. Another conjugate is ovalbumin (OVA), the main protein found in egg whites. OVA is soluble in polar reagents, making it a strategic carrier for antigens that are also polar.