Immunohistochemistry

IHC is usually used to determine the distribution and expression of 1 or 2 target molecules whereas IF staining is used for co-localization studies or for detection of multiple proteins on a section. IHC staining is permanent and does not fade like IF staining. In addition, tissue architecture and morphology are more readily apparent in IHC stained slides, making interpretation easier.

Both formalin-fixed paraffin embedded tissue (FFPE) or frozen (fixed or fresh) sections can be used. In fact, we have established a protocol that quenches autofluorescence when performing IF staining on FFPE tissue.

The Core facility routinely uses the enzyme horseradish peroxide combined with the DAB chromogen (in brown) with hematoxylin (blue) nuclear counterstain. We also offer chromogenic detection using the enzyme alkaline phosphatase combined with the Fast Red chromogen (in pink) for single or dual staining with DAB.

An antibody titer is the highest dilution of the antibody that results in a specific signal with minimal background or non-specific staining. The Core facility offers a complete antibody work up on all new antibodies, this includes titration of the antibody concentration on positive and negative control tissue sections using a variety of antigen retrieval methods.

The Core facility uses a polymer-based amplification system which has increased sensitivity compared to other detection methods, resulting in using a higher dilution of your primary antibody. Thus, it is highly recommended that all new antibodies be titered.

We do not supply any primary antibodies but can assist in selecting the appropriate antibody for your study. The Core facility supplies the majority of secondary and detection reagents for both IHC and IF staining.