Carl lsm710

4T1 cells were seeded onto coverslips in 24-well plates and cultured for 24 h. The next day, cells were treated with naked FAM-siRNA, PEI1.8k/FAM-siRNA or PCPP/FAM-siRNA for 4 h. For PCPP/FAM-siRNA, cells were incubated in pH 6.5 medium before nanoparticles were added. After washed with cold PBS, cells were fixed with 4% paraformaldehyde for 15 min. DAPI was subsequently added to stain the nuclei. Finally, the sample was observed and imaged on a confocal microscope (Carl Zeiss LSM 710, Carl Zeiss Microscopy GmbH, Germany). For endosomal/lysosomal escape investigation, cells were incubated with LysoTracker^® Red DND-99 at 37 ℃ for 1 h before fixed with 4% paraformaldehyde.
To verify the sialic acid receptor-mediated cellular uptake, cells were incubated with sialidase (50 mU mL^-1) for overnight prior to addition of PCPP/Cy3-siRNA. The sialidase catalyzes the hydrolysis of SA residues from glycoproteins and oligasaccharides of cell surfaces. Then the cells were incubated with FITC-SNA, MAA or WGA (SNA 1μg mL^-1, MAA 5μg mL^-1, WGA 5μg mL^-1) for 30 min, followed by CLSM analysis to detect the SA on cell surfaces. And the intracellular uptake by normal cells was used as a control.

The lung samples from the mice were immobilized with OCT (Sakura Finetek Europe B.V.) at −80°C for ≥24 h. The frozen samples were cut on a cryostat microtome, and the 7-µm sections were placed on polylysine-coated glass slides. The tissue sections were fixed in 4°C acetone and rehydrated in PBS. The slides were gently washed in PBS for three times and then blocked with 5% goat serum (cat. no. 16210-064; Gibco; Thermo Fisher Scientific, Inc.; 1:500 in PBS) at 37°C to reduce non-specific binding. After 30 min, the sections were washed with PBS and stained with Histone H3 (citrulline R2+R8+R17) antibody (cat. no. ab5103; Abcam; 1:300 diluted) (23 (link)) overnight at 4°C in the dark. Subsequently, the sections were gently washed in PBS and then stained with the fluorescent-conjugated secondary antibody [Goat anti-Rabbit IgG (H+L) Alexa Fluor^® 555 conjugate, cat. no. A-21428; Invitrogen; Thermo Fisher Scientific, Inc.] at 37°C for 60 min in the dark. After the unbound fluorescent antibody was removed, the sections were incubated with SYTOX Green diluted in PBS (1:2,000) at 37°C for 15 min. The sections were washed again, and then observed and recorded using confocal microscopy (magnification, ×100; CarlZeiss LSM710; Zeiss AG).

Fluorescence microscopy measurements were carried out in both epi- (Eclipse E600-POL, Nikon) and confocal (Carl Zeiss LSM710, Carl Zeiss) geometries. Texas Red and Alexa-Fluor 488 probes were fluorescently excited at appropriate wavelengths of He-Ne lasers and collected with appropriate excitation/emission filters (Ex: 590 nm, Em: 610 nm for Texas Red and Ex: 500 nm, Em: 526 nm for Alexa Fluor 488).