This protocol for in vivo immunofluorescence localization, or IVIL, assesses the in vivo biodistribution of diagnostic and therapeutic antibodies or antibody-based agents for cancer research, detection, and treatment. In contrast to conventional immunofluorescent staining where cellular expression of antigens is revealed ex vivo, the IVIL method elucidates how antibodies and antibody-based agents distribute in the living animal. This video will aid understanding of the overall workflow of the IVIL method.
It shows important details for successful reproduction of the protocol for other applications and antibodies. Observe mice from the desired cancer model for the appropriate tumor growth via palpation or caliper measurement before proceeding. Purify rabbit anti-mouse B7-H3 and rabbit IgG isotype control antibodies on a desalting column to remove preservatives and storage buffers following the manufacturer instructions.
Aliquot dosages of 33 micrograms of each antibody conjugate in individual microcentrifuge tubes. Following anesthetization as described in the text protocol, prepare for the tail vein inoculation of the antibody solutions. Disinfect the tail of the animal by wiping three times with an alcohol wipe.
Dilate the tail veins by warming with a heat pad for approximately 30 seconds. Avoid heating the entire animal. Using a 27-gauge tail vein catheter, insert the butterfly needle into one of the two lateral tail veins.
Visualize blood backflow into the catheter to ensure that the needle is properly placed so that the solutions will fully enter the animal versus being captured in the tail. Use a piece of surgical tape to carefully fix the tail with the inserted needle to the stage. Flush the catheter with 25 microliters of sterile phosphate-buffered saline.
Then, inject the antibody solution into the catheter using insulin syringes. Flush the catheter once again with 25 microliters of sterile PBS. Remove the needle from the tail, and apply pressure to stop any bleeding.
Perform humane euthanasia of the mouse as described in the text protocol, and lay the mouse in the supine position. Use surgical scissors and forceps to excise tumor tissues. Use forceps to grasp only the outer layer of skin between the set of mammary glands closest to the tail, and make a small incision with a pair of surgical scissors.
Introduce the closed scissors into the cut, and slowly open the tip to carefully separate the skin from the underlying abdominal wall membrane, keeping it intact. Make a vertical incision up the abdomen, continuing to separate the skin from the inner membrane. Between the third and fourth mammary glands, make a horizontal cut across the abdomen to allow retraction of the skin and visualization of the mammary glands.
Grasping each tumor or normal gland with forceps, carefully trim away the attached skin using surgical scissors. Place excised tissues into tissue disposable base molds that are prelabeled and filled with optimal cutting temperature embedding medium. Quickly freeze the molds by placement on dry ice.
In order to study off-target delivery, excise other tissues or organs of interest. Using a cryostat, section frozen tissue blocks at 10-micron thickness, and place adjacent sections onto prelabeled adhesion glass slides. Rinse frozen tissue slides with room temperature PBS for five minutes to remove the embedding medium.
Demarcate tissue sections with a hydrophobic barrier pen to reduce the volume of solutions needed during staining. Fix the tissue sections with 4%paraformaldehyde solution for five minutes. After rinsing the slides in PBS for five minutes, permeabilize tissue sections with 0.5%Triton X-100 in PBS for 15 minutes.
Rinse the slides again in PBS for five minutes. Block the tissues with PBS containing 3%weight per volume bovine serum albumin and 5%volume per volume goat serum for one hour at room temperature. After rinsing the slides in PBS for five minutes, incubate the sections with record-keeping primary antibodies such as a common nuclear, vascular, or cytoplasmic marker.
Here, rat anti-mouse CD31 is used at a one-to-100 dilution in the blocking solution. The slides with primary antibody are left overnight at four degrees Celsius, protected from dehydration on a slide tray. Following incubation, rinse the slides in PBS for five minutes three times.
Change the PBS each time. Incubate the slides with secondary antibodies to label the primary antibodies. For this application, visualize anti-B7-H3 antibody using Alexa Fluor 546 conjugated goat anti-rabbit antibody, and visualize CD31 with Alexa Fluor 488 goat anti-rat secondary antibody in blocking solution.
Protect the slides from light and dehydration on a slide tray for one hour at room temperature. After rinsing the slides in PBS as before, apply one drop of the mounting medium into the center of the tissue slice. Carefully place a coverslip, avoiding entrapment of air bubbles.
Seal the edges of the coverslip with clear nail polish, and allow to dry. Proceed with confocal microscopy imaging and quantitative image analysis as described in the text protocol. Representative confocal micrographs show the comparison of specific B7-H3 antibody-ICG conjugate and nonspecific isotype control antibody-ICG conjugate localization in murine mammary glands containing normal or carcinoma tissues.
Normal murine mammary glands from an animal intravenously injected with Iso-ICG or B7-H3-ICG and counterstained with CD31 show no staining of the antibody conjugates. Normal tissues were shown to have no expression of B7-H3 by standard ex vivo immunofluorescence staining. In invasive mammary tumors, B7-H3-ICG strongly binds to the vasculature, the first point of contact in vivo, and then is able to extravasate from the vasculature to heterogeneously stain the tumor epithelium.
Iso-ICG shows nonspecific accumulation within the tumor tissues. Standard ex vivo immunofluorescent staining shows uniform expression of the B7-H3 marker on epithelial and endothelial cells. Representative confocal micrographs show the in vivo immunofluorescence localization method to detect netrin-1 expression or isotype control expression in murine carcinoma and normal mammary glands.
In vivo immunofluorescence localization confirms epithelial signal for netrin-1 in MMTV-PyMT tumors but not in normal mammary glands. Furthermore, there is a strong netrin-1 signal on endothelial cells in breast tumors, as indicated by the colocalized yellow signal. The signal is significantly weaker in normal mammary glands.
The IVIL method will need to be optimized in terms of antibody dosage, tissue collection timing, and secondary antibody dilution for successful implementation. IVIL allows targeting of conformational epitopes that cannot be detected using standard immunofluorescence staining, which requires tissue processing. Also, healthy organs of tumor-bearing mice can be collected to assess off-target delivery of therapeutic antibodies.
With increased use of antibody-based therapeutics and contrast agents in cancer research and management, the IVIL method is highly applicable to pharmaceutical research and development. Researchers in cancer therapies, molecular imaging, inflammation, and other areas may find that the IVIL method provides useful information.
