IHC/IF/ISH

Mouse_islet_insulitisSingle 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, cytospins, and cell culture sections. Services include:

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
  • Animal and human block archives for assay controls

Immunolocalization:

  • IHC: single and multiple (polymer, HRP, AP, PE OPAL)
  • IF: secondary antibodies for a wide range of host species (rabbit, mouse, guinea pig, rat, goat, sheep) with multiple fluorochromes for multiplexing
  • ISH: RNAScope technology and others
  • Microscopy: Bright field, fluorescence, whole digital slide scans
  • Dako autostainer for high through put batch staining (up to 48 slides)
  • Immuno-EM available through the COM EM Core.

User provides primary antibody and sections on slides. A small selection of routine primary antibodies are stocked and these are included for free- please inquire.Extensive menu of routinely used antibodies:

  • Diabetes-related islet hormones, transcription factors, and cell developmental and regeneration markers (h, m, r)
  • Immunotyping mouse or human leukocytes
  • Proliferation: BrdU, Ki-67 (h,m,r), PCNA (h,m,r)
  • Apoptosis: TUNEL (h,m,r), cleaved caspase-3 (h, m)
  • Angiogenesis: CD31 (h,m), CD34 (h, m), Factor VIII (vWF, h, m)
  • Cancer: p53 (h), beta-catenin (h,m,r), focal adhesion kinase (h)
  • Others: Collagen (ECM, h, m), E-cadherin (m), GFAP (m), S100
Species: Human (h), Mouse (m), Rat (r)

For new antibody testing, please contact Dr. Ann Fu, Core Lab Manager, at 352-273-7749 or fudo@ufl.edu.

For initial testing of new antibodies, a series of antigen retrieval methods may be used on a known positive sample followed by 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 treatment control.

When an antibody lot is changed or an antibody has not been used for several months, 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 can not be reproduced, the antibody may undergo revalidation or an alternative antibody could be chosen. These options along with other possible approaches will 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. Control slides and probes can be purchased from the Core.Virus infected cell line (Dr. Ann Fu)

“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