Alexa 647 conjugated goat anti rat igg

Balb/c mice were orthotopically injected with 1 million of 4T1 cells in the mammary gland and after 3 days FAM-LinTT1-PS (1mg of polymer, 100μL) was intravenously injected. After 24 h, the animals were sacrificed and the tumor and organs were excised, fixed in 4% of paraformaldehyde, cryoprotected with 15% and 30% sucrose, frozen down with liquid nitrogen, and cryosectioned at 10 μm. Tissue sections were permeabilized using PBS 10 mM containing 0.2% Triton-X for 10 min, and blocked in PBS 10mM containing 0.05% Tween-20, 5% FBS, 5% BSA, and 5% goat serum (GE Healthcare, UK) for 1 h. The sections were immunostained at dilution 1/100 with anti-fluorescein rabbit IgG fraction (Thermo Fisher Scientific, MA, USA), rat anti-mouse CD31, biotin rat anti-mouse CD11b, (BD Biosciences, CA, USA), rat anti-mouse CD68, rat anti-mouse CD206 (Bio-Rad, USA), and anti-p32 rabbit polyclonal antibody (Millipore, Germany) as primary antibodies. As secondary antibodies, Alexa 488-conjugated goat anti-rabbit IgG and Alexa 647-conjugated goat anti-rat IgG (1/500, Invitrogen, Thermo Fisher Scientific, MA, USA) were used. The sections were counterstained with DAPI and examined by fluorescence confocal microscopy using Olympus FV1200MPE instrument. The images were processed and analyzed using the FV10-ASW 4.2 Viewer image software (Olympus, Germany) and the Image J software.

VE-cadherin in whole-mounted retinas was visualized as previously described 24 (link). Briefly, enucleated eyes were fixed with 4% paraformaldehyde for 45 min at room temperature and then with acetone for 3 min at -20°C. The fixed retinas were permeabilized for 4 h with 1.0% Triton X-100 in PBS and incubated with a monoclonal VE-cadherin antibody (1:200; BD Pharmingen, San Diego, CA, USA). The retinas were then probed with Alexa 647-conjugated goat anti-rat IgG (1:300; Invitrogen), and VE-cadherin in the superficial vessels of the retinas was visualized by confocal microscopy.
Actin filaments in whole-mounted retinas were determined as previously described 24 (link). Briefly, enucleated eyeballs were fixed with 4% paraformaldehyde overnight at 4°C. Following dissection in a tri-lobe configuration, the retinas were permeabilized with 1.0% Triton X-100 in PBS for 4 h and incubated with Alexa Fluor 488 phalloidin (1:200; Molecular Probes, Eugene, OR) for 2 h at room temperature. The superficial vessels in the stained whole-mounted retinas were visualized by confocal microscopy.

4T1 and MCF10CA1a cells were detached from the culture plate with enzyme-free cell dissociation buffer (Gibco, Life Technologies, UK). The cells (10⁵ cells) were incubated with 10μg/mL of the in-house generated rabbit polyclonal p32 antibody in blocking buffer containing 1% of BSA, 1% FBS, and 1% of goat serum in PBS at room temperature for 1 h. The cells were washed, and incubated with Alexa 647-conjugated goat anti-rat IgG (1/1000, Invitrogen, Thermo Fisher Scientific, MA, USA) in blocking buffer at room temperature for 30 min. After washes, the cell surface p32 expression was analyzed by flow cytometry (Accuri, BD Biosciences, CA, USA).