Immunopathology Lab: Research and Development

"Immunohistochemistry is technically complex, and no aspect of this complexity can be ignored, from the moment of collecting the specimen to issuance of the final report." Taylor, CR., Arch Pathol Lab Med

"There is no one universal fixative for IHC. Each new antibody must be assessed individually."

The Immunopathology lab provides research support and developmental services for immunolocalization assays for both proteins and nucleic acids. Expertise is available for immunoperoxidase, immunofluorescence, in situ hybridization, microscopy, morphometric analysis, and many other techniques used for microscopy-based analyses.

Staff:
Dr. Ann Fu, Lab Manager
Dr. Li Zhang, Sr. Biological Scientist
Amy Wright, MS, Sr. Biological Scientist
Cathy Sun, Sr. Laboratory Technician

Room: D11-39, D11-50
P: (352) 273-7754, 273-7755
F: (352) 273-7753
E : molecular@pathology.ufl.edu

Image analysis mouse islet	Double staining with co-localization (yellow)	Rat Y and 12 chromosomes	Human tumor p53

Services:

Fee for service assays (formalin fixed paraffin embedded):

• Reporters: GFP, RFP, luciferase, c-myc
• Neuroendocrine hormones: insulin (h,m,r,pr), gastrin, somatostatin, glucagon, ghrelin, pancreatic polypeptide, serotonin, many others
• Immunotyping (h,m,r): CD20, CD45, B220 (B cells); CD3,4,8 (T cells); F4/80/CD68 (macrophages); many others
• Proliferation markers: BrdU, Ki-67 (h,m,r), PCNA (h,m,r)
• Apoptosis: TUNEL, cleaved caspase-3 (h, m)
• Vasculature (h, m, r): CD31, CD34, Factor VIII (von Willebrand factor), collagen, fiobronectin, smooth muscle actin
• Cancer: p53 (h), beta-catenin (h,m,r), focal adhesion kinase (h)
• FISH: commerical probes (h,m,r)
• Others- please inquire as many more have been developed for paraffin and fixed and fresh frozen samples
  • Species: Human (h), Mouse (m), Rat (r), Primate (pr)

Immunoenzyme (peroxidase, alkaline phosphatase) and immunofluorescence assays are available with several chromagens, fluorchromes, and counterstain combinations. Immunoperoxidase detection using the polymer-HRP system (Biocare Medical) is our most frequently used technique with DAB (brown) as chromagen. To work up a new antibody, a series of antigen retrieval methods are tested including heat-based (citrate pH 6.0, EDTA solutions, Trilogy), and enzymatic digestion (trypsin). Positive and negative control slides are included with each run. These slides can be provided by the investigator or may be obtained from the Core's control archive. Negative control slides include one or more of the following conditions: no primary, substitution of primary with host species IgG or normal serum or a treatment control in animals or cells.

For developing new antibodies, the investigator should be prepared to discuss teh available primary antibody with product sheet and suitable positive control samples. Data from Western blot analysis or publications documenting specificity and sensitivity are useful. Please contact Dr. Fu for setting up a consultation to discuss your project.

Equipment:

• Autostainer plus with Seymour glass slide labeling system (Dako)
• In situ hybridization oven (Dako)
• Decloaker (Biocare), water baths, steamer, and microwave for antigen retrieval
• *Zeiss Axioskop Plus motorized microscope with Axiocam HR color camera
  (Filter sets: #01, YFP , #10, #14 , #20, FITC/Texas Red (Omega XF1051, XF3057))
• *Axiovision (Zeiss), Metamorph, and Spectrum (Aperio) image analysis software
  • *Use is limited to those using Core histology or immunopathology services
• Aperio ScanScope CS
• Confocal microscopes are available through ICRB and MBI Cores

Useful IHC Links:
Antibody search
Preparing antigen retrieval and paraformaldehyde solutions
BrdU-injection and IHC
NordiQC IHC techniques

Frozen tissue sectioning and staining-BD
Immunohistochemistry Links
Protocols on-line IHC
ISH with Dig-riboprobes | FISH protocols
Optimizing IHC protocols
DNA/RNA from tissue sections (Qiagen)

Great Mouse IHC Protocols:
Rodent IHC | Hematopoietic antigens in paraffin-embedded tissues (NIAID)
Antibodies optimized for mouse (NCIFCRF) | Jackson Labs | NIEHS
Columbia University | Beta Cell Consortium Antibody Core