Source: Wischhusen, J. C., et al. In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research. J. Vis. Exp. (2019).
This video demonstrates an in vivo immunofluorescence localization (IVIL) technique to study the biodistribution of tumor-targeting antibodies in a murine model of breast cancer. Upon the intravenous injection of a tumor-specific antibody in the mouse, the tumor is surgically isolated, and the localization of the antibody within the tumor tissue is confirmed via ex vivo immunostaining.
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Transgenic mouse model of breast cancer development
2. Intravenous injection of specific and nonspecific antibody agents
3. Collection and preparation of target tumor tissues
4. Ex vivo staining protocol
NOTE: For the quantitative comparison between fluorescence microscopy images, all slides are stained at the same time with the same prepared solutions.
5. Confocal microscopy imaging and quantitative image analysis
NOTE: Preparing the confocal microscope and imaging parameters will depend on the confocal system used. The microscope used here was purchased commercially (e.g., Zeiss LSM 510 Meta system) and the associated acquisition software was used (e.g., Zen 2009). However, many of these steps will apply to any confocal microscope and assume basic confocal microscopy knowledge.
The authors have nothing to disclose.
Animal Model | |||
FVB/N-Tg(MMTV-PyMT)634Mul/J | The Jackson Laboratory | 2374 | Females, 4-6 weeks of age |
Animal Handling Supplies | |||
27G Catheter | VisualSonics | Please call to order | Vevo MicroMarker Tail Vein Access Cannulation Kit |
Alcohol Wipes | Fisher Scientific | 22-246073 | |
Gauze Sponges (4" x 4" 16 Ply) | Cardinal Health | 2913 | |
Heat Lamp | Morganville Scientific | HL0100 | |
Isoflurane | Henry Schein Animal Health | 29404 | |
Ophthalmic Ointment | Fisher Scientific | NC0490117 | |
Surgical Tape | 3M | 1530-1 | |
Tissue Collection | |||
Disposable Base Molds | Fisher Scientific | 22-363-556 | |
Optimal Cutting Temperature (OCT) Medium | Fisher Scientific | 23-730-571 | |
Surgical London Forceps | Fine Science Tools | 11080-02 | |
Surgical Scissors | Fine Science Tools | 14084-08 | |
Antibodies | |||
AlexaFluor-488 goat anti-rat IgG | Life Technologies | A-11006 | |
AlexaFluor-546 goat anti-rabbit IgG | Life Technologies | A-11010 | |
AlexaFluor-594 goat anti-human IgG | Life Technologies | A11014 | |
Human IgG Isotype Control | Novus Biologicals | NBP1-97043 | |
Humanized anti-netrin-1 antibody | Netris Pharma | contact@netrispharma.com | |
Rabbit anti-Mouse CD276 (B7-H3) | Abcam | ab134161 | EPNCIR122 Clone |
Rat anti-Mouse CD31 | BD Biosciences | 550274 | MEC 13.3 Clone |
Reagents | |||
Bovine Serum Albumin (BSA) | Sigma-Aldrich | A2153-50G | |
Clear Nail Polish | Any local drug store | ||
Indocyanine Green – NHS | Intrace Medical | ICG-NHS ester | |
Mounting Medium | ThermoFisher Scientific | TA-006-FM | |
Normal Goat Serum | Fisher Scientific | ICN19135680 | |
Paraformaldehyde (PFA) | Fisher Scientific | AAJ19943K2 | |
Sterile Phosphate Buffered Saline (PBS) | ThermoFisher Scientific | 14190250 | |
Triton-X 100 | Sigma-Aldrich | T8787 | |
Supplies | |||
Adhesion Glass Slides | VWR | 48311-703 | |
Desalting Columns | Fisher Scientific | 45-000-148 | |
Glass Cover Slips | Fisher Scientific | 12-544G | |
Hydrophobic Barrier Pen | Ted Pella | 22311 | |
Microcentrifuge Tubes | Fisher Scientific | 05-402-25 | |
Slide Staining Tray | VWR | 87000-136 | |
Software | |||
FIJI | LOCI, UW-Madison. | Version 4.0 | https://fiji.sc/ |