Single and multiplex immunolocalization protocols for protein and mRNA are available. Staff members have extensive experience with > 500 primary antibodies for animal and human fixed and frozen sections, cytospins, and cell culture.
Consultation and Training
- Project planning, tissue selection, and experimental design.
- Protocol development and validation
- Sharing of antibody-specific protocols
- Procuring antibodies or ISH/FISH probes and testing
- Animal and human block archives for assay controls
- IHC: single and multiple (polymer, HRP, AP, OPAL)
- IF/Multiplex IF: direct and indirect with secondary antibodies provided for a wide range of primary antibody host species (rabbit, mouse, guinea pig, rat, goat, sheep).
- ISH: ACD RNAScope technology and others
- Microscopy: Brightfield, fluorescence, laser microdissection, whole slide scans
- Leica Bond autostainer for batch staining (up to 36 slides)
User provides primary antibody and slides. A small selection of free primary antibodies is stocked. For new antibody testing, please contact Dr. Ann Fu, Core Lab Manager, at 352-273-7749 or email@example.com.
For initial testing of new antibodies, different antigen retrieval methods may be tested using a known positive sample before testing on your sample of interest. Serial dilutions of the antisera are done to achieve the most specific and sensitive titer. Negative controls include the following: substitution of primary antibody with host species IgG, antisera peptide pre-absorption, and/or experimental tissue control.
When an antibody lot is changed or an antibody has not been used for several months/years, verification is performed by testing the last known set of validated conditions on similar samples. Once verified, experimental samples can be run without further validation. If antibody performance cannot be reproduced, the antibody may undergo revalidation or an alternative antibody could be chosen. These options along with other possible approaches can be discussed during the initial consultation.
In situ hybridization (ISH/FISH)
DNA and RNA nucleic acid detection services are available for users providing probe and slides. ACD RNAScope probes are recommended for mRNA detection by in situ hybridization. Assay optimization is performed using varying sample digestion temperatures and times. Positive and negative tissue control slides and probes can be purchased from the Core.
“There is no one universal fixative for IHC… Immunohistochemistry is technically complex, and no aspect of this complexity can be ignored, from the moment of collecting the specimen…”
– Taylor, CR., Arch Pathol Lab Med