Immunopathology Lab: Research and Development

The Immunopathology lab provides immunostaining services for established antibodies as well as optimization of new antibodies. Expertise and equipment are available for immunoperoxidase, immunofluorescence, in situ hybridization, FISH, microscopy, image analysis, and many other techniques used for microscopy-based immunolocalization.

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

Rooms: D11-39, D11-50
P: (352) 273-7754, 273-7755
F: (352) 273-7753

Insulin image analysis mouse islet	Double IF staining with co-localization (yellow)	FISH: Rat Y and 12 chromosomes	IF: Human tumor p53

Services:

• 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 (See scope filters), and counterstain combinations. Immunoperoxidase detection using the polymer-HRP system (Biocare Medical) is our most frequently used technique with DAB (brown) as chromagen and hemaoxylin counterstain. Double and triple staining assays are also available.

For developing new antibodies, the investigator should provide the primary antibody and blocks/slides and be prepared to discuss the suitability of the antibody based on its product sheet and/or available publications along with suitable controls.

To work up an untested 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 archives. Negative control slides include one or more of the following conditions: no primary, substitution of primary with host species IgG or normal serum, peptide absorption, and treatment control.

Results with new antibodies can not be guaranteed when samples are not fixed properly and/or processed in other facilities. Fees will be assessed based on work done. Please note the following:

"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

Equipment:

• ThermoShandon Cytospin 4
• Dako Autostainer plus and Seymour slide labeling system
• Dako in situ hybridizer
• Biocare Decloaker, water baths, steamers, and microwaves for antigen retrieval
• *Zeiss Axioskop Plus motorized microscope with Axiocam HR color camera and Axiovision 4.7
  (Filters: #01, YFP , #10, #14 , #20, FITC/Texas Red, others)
• *Axiovision 4.7, Metamorph, and Spectrum (Aperio) image analysis software, Adobe Photoshop CS
  • *Use is limited to those using Core services and requires training before independent use
• Aperio ScanScope CS
• Confocal microscopes are available through other UF Cores (ICBR, MBI)
• Laser capture microdissection is available through the Moffitt Analytic Microscopy Core.

IHC Links:
Antibody search | Human Protein Atlas

Preparing antigen retrieval and paraformaldehyde solutions
BrdU-injection and IHC
NordiQC IHC techniques

Frozen tissue sectioning and staining-BD | Dako immunostaining methods (4th ed)
Immunohistochemistry Links
Protocols on-line IHC
ISH with Dig-riboprobes | FISH protocols
Optimizing IHC protocols
DNA/RNA from tissue sections (Qiagen)

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