Zenon rabbit igg labeling kit

U87MG and H4 cells (1 × 10⁴ cells per well) were cultured in 8-well chamber slides (Lab-Tek). Before staining, the cells were starved, activated, or treated with AZD8055 for 24 h. For the knockdown conditions, cells were incubated with siRICTOR, siMAPKAP1, FANA-GSN, and scramble controls for 48 h prior to the steps. Cells were fixed with 4% paraformaldehyde, lysed with 0.2% Triton X buffer, blocked with 1% BSA, and incubated with primary antibodies (anti-RICTOR, anti-MAPKAP1, anti-GSN, anti-β-tubulin (Abcam), anti-mTOR, anti-VIM (Cell Signaling Technologies), anti-FLNA (Millipore), Alexa Fluor 647 Anti-MYH9 (Abcam), Alexa Fluor 488-phalloidin (Invitrogen)) overnight at 4 °C. Samples were stained with secondary antibodies, including Alexa Fluor 488, Alexa Fluor 594, Alexa Fluor 647 Anti-Rabbit antibodies, and Alexa Fluor 568 Anti-mouse antibody. A Zenon Rabbit IgG labeling kit (Invitrogen) was used to directly conjugate to primary antibodies when necessary. Nuclei of cells were stained with DAPI. Slides were mounted with ProLong anti-fade mountant (Invitrogen). Cells were visualized by an LSM800 with an Airyscan confocal microscope (Zeiss).

A flow cytometric analysis was performed using the FACSCalibur and LSRFortessa platforms (BD Biosciences, Tokyo, Japan). For Ki-67 and 7-amino-actinomycinD (7-AAD) staining, the FOXP3 Staining Buffer Set (eBioscience) was used. Antibodies conjugated with FITC, PerCP or APC were used. Anti-human CD14 (clone MφP9), anti-human CD45 (clone 2D1) antibodies and 7-AAD were purchased from BD Biosciences. Anti-human CD1a (clone HI149), anti-human Ki-67 (clone Ki-67) antibodies and Fixable Viability Dye (FVD520) were obtained from eBioscience. The cells were washed and stained. For intracellular staining of survivin and its splice variants, normal rabbit IgG (Santa Cruz Biotechnology, TX, USA), anti-survivin (rabbit polyclonal, R&D Systems), anti-survivin-ΔEx3 (rabbit polyclonal, Abcam), and anti-survivin-2B (rabbit polyclonal, Abcam) were labeled using the Zenon Rabbit IgG Labeling Kit (Alexa Fluor 647, Invitrogen). The FOXP3 Staining Buffer Set (eBioscience) was used for fixation and permeabilization. A survivin-WT-specific antibody was not available, therefore, the mean fluorescence intensity (MFI) of the estimated survivin-WT expression level was calculated from other MFI parameters as follows: (survivin-WT) = (survivin (total) – normal IgG) – (survivin-ΔEx3 – normal IgG) – (survivin–2B – normal IgG).

Drosophila embryos were stained, essentially as previously described23 (link). Basically, 0–1-h Drosophila embryos were collected and washed with cold water. The chorions were removed by breech, and the remaining embryos were suspended in buffer containing 70 mM NaCl and 0.03% Triton X-100. After devitalizing, embryos were then incubated four times for 30 min each in 10% bovine serum albumin in PBS. Subsequently, the embryos were incubated with Alexa 488-labeled anti-SMARCAD1 antibodies that had been labeled overnight at 4 °C using a Zenon Rabbit IgG labeling kit (Molecular Probes). The embryos were washed and then incubated with Hoechst 33342 dye for visualizing cellular DNA. The embryos were washed, and confocal images were collected with an Olympus IX81 microscope.