Goat anti rat igg

Immunofluorescence method was used to investigate the tumor-penetrating ability of Fe₃O₄@SiO₂@mSiO₂/DiO-(Gd-DTPA)-PEG-RGE NPs. The Fe₃O₄@SiO₂@mSiO₂/DiO-(Gd-DTPA)-PEG-RGE NPs were i.v. injected to U87MG tumor-bearing mice (n = 3). The mice were sacrificed 8 h post-injection, and the tumor tissues and main organs (including heart, liver, spleen, lung, and kidney) were collected, fixed, dehydrated, and frozen in acetone/dry ice mixture. The frozen samples were further cut to sections (10 μm) with a cryostat (CM3050 S, Leica, Germany). The sections were stained with rat anti-mouse CD31 (1:10, R&D Systems), followed by rhodamine labeled goat anti-rat IgG (1:100, Santa Cruz) and Hochest33342, and then observed using a confocal laser scanning microscope (CLSM, TCS SP5, Leica, Germany).

One microgram of vesicular proteins was separated by SDS-PAGE and then transferred to a 0.2 µm polyvinylidene difluoride membrane. The membrane was blocked with 5% non-fat milk or 3% skim milk in Tris-buffered saline with 0.05% Tween-20, incubated with primary antibody followed by secondary antibody conjugated with horseradish peroxidase, and subjected to the enhanced chemiluminescence. The membrane was washed three times by Tris-buffered saline with 0.05% Tween-20 after each incubation. Goat anti-actin (1:1000 dilution, SC-1616), rabbit anti-GAPDH (1:1000 dilution, SC-257,780), goat anti-mouse IgG (1:5000 dilution, SC-516,102), goat anti-rabbit IgG (1:5000 dilution, SC-2004), donkey anti-goat IgG (1:5000 dilution, SC-2020) and goat anti-rat IgG (1:5000 dilution, SC-2006) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-CD81 (1:1000 dilution, 555,675) and mouse anti-calnexin (1:1000 dilution, 610,523) antibodies were from BD Biosciences (San Diego, CA). Goat anti-ICAM1 (1:1000 dilution, BBA17) antibody was obtained from R&D Systems (Abingdon, UK). Mouse anti-60 S ribosomal protein L14 (RPL14) (1:1000 dilution, ab89095) antibody was from Abcam (Cambridge, MA). Mouse anti-tubulin (1:1000 dilution, T6074) antibody was from Sigma (St. Louis, MO). Rabbit anti-H2B (1:1000 dilution, 07–371) antibody was obtained from Millipore (Darmstadt, Germany).

Vero cells infected with EV71 H3-TY were incubated in a 96-well plate at 35°C in 5% CO₂. When CPEs were observed (at least 25%), cells were fixed with precooled acetone. Antibodies diluted to 1:100 in PBS were added to the plates blocked with 1% BSA in PBS, and incubated overnight at 4°C. After washing five times with PBS containing 0.01% Tween-20 (PBS-T), FITC-conjugated goat anti-mouse IgG, goat anti-rat IgG, or goat anti-rabbit IgG (diluted 1:50 in PBS-T; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were added and the plates were incubated for 1 h at 37°C. After washing five times with PBS-T, fluorescent images were visualized and captured using a DM BL2 fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany).