| |
|
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 2000; 124:945.
The Immunopathology lab provides research support and developmental services for immunolocalization assays for proteins and nucleic acids. Expertise is available for immunoperoxidase, immunofluorescence, in situ hybridization,
microscopy, image processing, and many other techniques used for microscopy-based analyses.
The Immunopathology staff prioritize their time to provide research services for program project grants (PIs: Drs. Mark Atkinson, Barry Byrne, and Nicholas Muzyczka) and are available for a limited number of fee-for-service projects. Please contact Dr. Fu regarding availability. If we are unable to provide IHC services at the time, we can provide hands-on training or protocols and can recommend the COM Electron Microscopy Core (Dr. Jill Verlander, Director) for fee-for-service IHC.
Staff:
Dr. Ann Fu, Lab Manager 
Dr. Li Zhang, Sr. Biological Scientist
Amy Wright, MS, Sr. Biological Scientist
Cathy Sun, Sr. Laboratory Technician
Mandy King, BS, Lab Assistant
Room: D11-39, D11-32
P: (352) 273-7754, 273-7755
F: (352) 273-7753
E
: molecular@pathology.ufl.edu
|
|
|
|
Image analysis mouse islet |
Co-localization (yellow) |
Services:
Routine and developmental immunopathology assays for frozen and paraffin samples.
Consultation on project design to optimize antigen stability, and thereby detection by immunolocalization, is encouraged before the tissues are harvested.
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, others
- Immunotyping (h,m,r): CD45, B220, CD3,4, 8, F4/80/CD68, many others
- Proliferation markers: BrdU, Ki-67 (h,m,r), PCNA (h,m,r)
- Apoptosis: TUNEL (mouse pancreas and kidney), cleaved caspase-3 (h, m)
- Endothelium (h, m, r): CD31, CD34, Factor VIII (von Willebrand factor)
- Cancer-related: p53 (h), beta-catenin (h,m,r), focal adhesion kinase (h)
- FISH: X,Y chromosomes (h,m), others when submitter provides probe
- Others- please inquire
as many more have been developed for paraffin and fixed and fresh frozen samples
Immunoenzyme (peroxidase, alkaline phosphatase) and immunofluorescence assays are available with several chromagen, fluorchromes, and counterstain combinations. Immunoperoxidase detection using the polymer-HRP system (Biocare Medical) is our most frequently used technique with cDAB (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 at low and high pH, Trilogy (Cell Marque, CMX833)) and enzymatic digestion(trypsin, pepsin). 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 or substitution of primary with host species IgG or an animal treatment control (PBS).
Optimizing IHC protocols
For developing new antibodies, the investigator should be prepared to provide positive control material (tissues or cell lines) and the primary antibody along with its product sheet. Data from Western blot analysis or publications documenting specificity and sensitivity are very useful when available.
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 dual (Omega XF1051, XF3057))
- *Axiovision and Metamorph image analysis software
- *Use is limited to MPC staff and labs with grants supporting staff in the facility
- Aperio ScanScope CS
- Refrigerators and freezers (-20 and -80) and other molecular biology equipment
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
FISH protocols
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 |
