Gattoretti Lab Protocols

 

Giorgio Cattoretti, MD

Columbia University, Institute for Cancer Genetics, New York

 

Mouse Pathology

• Processing mouse tissue

• Analysis of murine BM

• Removal of lymphoid organs

• Cell dissociation

• Harvesting peritoneal cells

• Nomenclature

• Submission forms

• Keep track of mice

Immunohistochemistry/Immunofluorescence

• General principles

• specimens

• FAQ

• Immunohistochemistry (Antigen Retrieval)

• DNAse post-treatment for nuclear antigens

• Double Immunohistochemistry

• Mouse-on-Mouse easy IHC (Ig isotype staining)

• Double Immunofluorescence stain

• BCL6 / PNA Immunohistochemistry

• AlkPhos with NBT/BCIP

• EBER in situ (for double staining)

• Antigen-specific staining

• Cytospin preparation

• slides storage

• Cell block preparation

• Tissue Micro Array Analysis Tool

• Blocking non-specific staining

Flow Cytometry

• Hemacytometer counting

• RBC lysis method
• Intracellular staining for FACS
• Nuclear BCL6 and cell cycle
• Nuclear/Cyto flow cytochemistry
• Annexin V-Pe 7-AAD protocol
• BrdU + PI staining
• Whole blood staining
• TUNEL + Nuclear staining
• Cell staining for FDG analysis
• Using polyclonals for flow
• A typical mouse FCM analysis

Reagents and Solutions

• AnnV buffer
• RBC Lysing solution
• HRP development solution (AEC)
• AP Developing solution
• Blocking solutions
• Endogenous peroxidase blocking solution
• Non-specific antibody background blocking solution: swine serum
• Non-specific antibody background blocking solution: milk
• Enzymatic antigen retrieval solution
• Unconjugated, biotin- and fluorochrome-conjugated antibody (primary) solutions
• Unconjugated, biotin- and fluorochrome-conjugated antibody (secondary) solutions
• Enzyme-conjugated secondary antibody solutions

Anti-Mouse Antibody Staining Gallery