An In Vivo Immunofluorescence Localization Method to Study Antibody Biodistribution

Abstract

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.

Protocol

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

1. Observe mice from the desired cancer model for the appropriate tumor growth via palpation or caliper measurement before proceeding.
   NOTE: Here, the transgenic murine model of breast cancer development (FVB/N-Tg(MMTV-PyMT)634Mul/J) (MMTV-PyMT) was used. These animals spontaneously develop invasive breast carcinomas between 6 and 12 weeks of age in each mammary gland. Normal mammary gl....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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/