Alexa 488 conjugated goat anti rabbit igg antibody

Six rats of each group were selected at random for immunofluorescence. After collection, tissues of were fixed in 4% paraformaldehyde and embedded in paraffin. Blocks were sectioned (5 μm), mounted on slides, then deparaffinized and rehydrated by successive incubations in xylene, 100% ethanol, 95% ethanol, 80% ethanol, 70% ethanol, and phosphate buffer saline (PBS). Custom anti-occludin antibodies (Abcam, Cambridge, MA, USA) were applied overnight at 4°C, followed by an Alexa 488–conjugated goat anti-rabbit IgG antibody for 60 min (Abcam, Cambridge, MA, USA). The slides were washed extensively and stained with DAPI (4’6’-diamidino-2-phenylindole) (Abcam, Cambridge, MA, USA). Images were obtained (Zeiss LSM 780 laser confocal microscopy) at excitation wavelengths of 390 nm and 500 nm; emission was detected at 440 and 580 nm. All the stainings were performed in duplicate in non-serial distant sections, and analyzed in a double-blind manner by two different experienced investigators.

Freshly isolated type II cells were plated on a plastic chamber slide (Lab-Tek II chamber slides, Nalge Nunc International, Naperville, IL). After overnight culture, the cells were infected with adenovirus (Ad-lacZ or Ad-Rab³8) at MOI = 2 to reduce expression level of Rab³8 protein and facilitate identification of co-localization with SP-B. The cells were further cultured for 24 h and washed twice with PBS. The cells were fixed with 4% paraformaldehyde for 10 min and followed with 100% acetone for 30 s. The fixed cells were blocked with 25% normal goat serum, incubated with a primary antibody (anti-Rab³8 and anti-SP-B), followed by a secondary antibody (Alexa 488-conjugated goat anti-rabbit IgG antibody and Alexa 594-conjugated goat anti-mouse IgG antibody) (Abcam, Tokyo, Japan). The slides were mounted with an antifade solution containing DAPI (S7113, Millipore, Temecula, CA). The immunofluorescence images were acquired using a fluorescence microscope (Keyence Biorevo BX-9000, Osaka, Japan).

MCF7, T47D and HC11 cells (5 × 10⁵ cells) were seeded on glass coverslips in a 24-well plate. After 24 h, PS were added to the cells at a concentration of 0.5 mg/mL and incubated for 1 h or 24 h. The spheroids were grown for 7 days after which PS were added at the same concentration as cell uptake. The cells or spheroids were washed with PBS, fixed with 4% of paraformaldehyde in PBS, immuno-stained with Alexa 488-conjugated goat anti-rabbit IgG antibody (Abcam, USA), stained with propidium iodide, mounted and imaged with fluorescence confocal microscopy (Zeiss LSM 510). The images were processed and analyzed using the Image J software.