Anti p32 rabbit polyclonal antibody

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.

4T1 cells (5x10⁵) were seeded on glass coverslips in a 24-well plate and the next day incubated with ATTO550-labeled polymersomes (0.5 mg polymer/mL) at 37°C for 1 h. Cells were washed with PBS, fixed with 4% paraformaldehyde, permeabilized with 0.5% saponin, and blocked for 1 h with 1% BSA, 1% goat serum, 0.3M of glycine, and 0.05% of Tween-20 in PBS. Cells were then stained for p32 protein using anti-p32 rabbit polyclonal antibody (1/500, Millipore, Germany) and Alexa Fluor 488 goat anti-rabbit IgG (1/1000, Abcam, UK) as a secondary antibody, and counterstained with 1 μg/mL of DAPI. Cells were examined under the confocal laser scanning microscope (LSM 510 Meta, Zeiss, Germany) equipped with a 63x oil objective lens (1.4 NA). Images were acquired sequentially to avoid cross-talk using excitation wavelengths 405, 488, and 561 nm. Transmission images were collected and overlaid by Zeiss Zen software.