Anti fluorescence quencher

Each cell crawl was fixed in 4% paraformaldehyde for 15min. To remove the 4% paraformaldehyde, crawl pieces were immersed in PBS for 5 min × three times. About 0.1% triton X-100 (Beyotime, China, ST795) was added to the cells until the cells were completely covered and incubated at room temperature for 30 min. To remove 0.1% triton X-100, cell crawl pieces were submerged in PBS for 5 min × three times. Anti-stainTM488 Fluorescent Phalloidin (Solarbio, China, CA1610) was dripped, diluted with PBS at 1:100, and then dripped until the cells were completely covered for incubation. The cell crawl pieces were immersed in PBS for 5 min × three times to washout primary antibody. DAPI (Aladdin, China, d106471-5 mg) was added dropwise to completely cover the cells to counterstain the nuclei. The cell crawl pieces were immersed in PBS for 5 min × 3 times to remove DAPI. Adhesive tip drops were added half a drop of antifluorescence quencher (Solarbio, China, s2100) on the slide, cell crawl pieces were buckled onto the slide with antifluorescence quencher drop. The staining effect was observed under a fluorescence microscope.20 (link)

Changes in the expression of recep tor CD44 were evaluated by an immunofluorescent assay and confocal laser scanning microscopy (Leica SPE, Germany). Experimental groups included sCS NPs-HA (amounts of Cy3-labeled siRNA was 0.52 μg), and the cells cultured in the RPMI 1640 medium were the control group. A549 cells were cultured on glass slides in 24-well plates for 24 hrs, followed by their incubation with the medium with or without sCS NPs-HA. Samples were collected at 4, 8, 16, 24, and 48 hrs and fixed in 4% paraformaldehyde for 15 min, followed by their permeabilization with 0.1% Triton X-100 for 10 min and blocking with 5% BSA for 30 mins. The slides were incubated overnight with a mouse polyclonal anti-CD44 antibody (CST, Beverly, MA, USA) at 4°C, followed by treatment with fluorescein isothiocyanate–conjugated secondary antibodies (mouse anti-rabbit fluorescent secondary antibody) (Abcam, Cambridge, UK) for 1 hr at room temperature. After a wash with PBS, nuclear staining was performed by the incubation of samples with DAPI (0.5 mg/mL) for 5 mins. The slides were washed once with PBS for 5 mins and sealed with an antifluorescence quencher (Solarbio, Beijing, China). The samples were examined under the confocal laser scanning microscope. At the same time, cells were collected at different time intervals and PCR experiments were performed to analyze the changes of CD44.

To assess phagocytosis visually, K7M2 and RAW264.7 cells were co-cultured as aforementioned. The co-cultured cells were added to a 24-well plate with round coverslips and cultured for 48 h. Cells that had grown on the coverslips were fixed using 4% formaldehyde at room temperature for 30 min, and counterstained using 10 µg/ml DAPI (Beijing Solarbio Science & Technology Co., Ltd.), for 10 min, followed by mounting in anti-fluorescence quencher (Beijing Solarbio Science & Technology Co., Ltd.). Finally, images were obtained by laser confocal microscopy (×63 magnification) (Leica GmbH).