Cy3 conjugated anti rabbit igg

Expression of Runx1t1 in BV2 microglial cells was inhibited using specific siRNA (Silencer siRNA transfection, Cat No. 4390771, Ambion, Inc. USA). Pre-designed Runx1t1 siRNA and Scrambled siRNA were obtained from Ambion (Table 1). The siRNA transfection in microglial cells was performed according to the manufacturer’s instruction. BV2 microglial cells were resuspended in OPTIMEM medium at a concentration of 1×10⁵ cells/ml. Transfection complexes were prepared by mixing RNAiMAX Lipofectamine transfection agent (Cat No. 13778-150, Ambion) and siRNA (20 nM) in OPTIMEM Serum-free Medium (Invitrogen Life Technology, USA ). Microglial cells were then mixed with transfection complexes and incubated for 24 h in 6-well (2×10⁵ cells/well) or 24-well (4×10⁴ cells/well) plates.
Runx1t1 protein expression in microglial cells was analyzed by immunofluorescence (n = 3). Transfected BV2 microglial cells were fixed with 4% paraformaldehyde in phosphate buffer for 30 min at room temperature. Following incubation in normal goat serum, all coverslips with adherent cells were incubated in rabbit anti-Runx1t1 (1∶100, Cat.No.sc-28693, Santa Cruz, USA) at 4°C overnight. Subsequently, the cells were washed with PBS and incubated with FITC-conjugated lectin and Cy3-conjugated anti-rabbit IgG (1∶200, Sigma) for 1 h. Cells were finally counterstained with DAPI (1 µg/ml, Invitrogen, USA).

Anti-phospho Smad2 (pSmad; Cat#AB3849), anti-Ki-67 (Cat# AB9260), HRP-conjugated anti-rabbit IgG (Cat# 12–348), and anti-vascular endothelial growth factor (VEGF; Cat# 05–443) were purchased from Millipore (Billerica, MA). Anti-caspase-3 antibody (Cat# 9661) was from Cell Signaling Technology (Danvers, MA). Anti-CD68 antibody (Cat# 6A324) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti- hypoxia-inducible factor 1-alpha (HIF-1α; Cat#NB-100-449) was purchased from Novus Biologicals (Littleton, CO). Anti-occludin (Cat#331500) and anti-ZO-1 (Cat#617300) antibodies were purchased from Invitrogen. Anti-granulocyte receptor-1 (Gr1; Cat# MBS520313) was from MyBioSource (San Diego, CA). Horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Cat# A4116), Cy3-conjugated anti-rabbit IgG (Cat# C2306) and anti-β-actin (Cat# A5441) antibodies were purchased from Sigma-Aldrich. Anti-myeloperoxidase (MPO) (Cat#DOM00001G), anti-claudin-3 (Cldn3; Cat# 341700), AlexaFlour 488-conjugated anti-mouse IgG (Cat# A11029), AlexaFluor 488-phalloidin (Cat# A12379), AlexaFluor 546-phalloidin (Cat# A22283), and anti-TNFα (Cat# AMC3012) antibodies were purchased from Thermo Fisher Scientific.

Mice were anesthetized, killed and inner-ear tissues were removed. The cochleae were further dissected and fixed in 4% paraformaldehyde”. Immunofluorescence staining with antibody against cleaved Caspase-3 (C-Casp3, rabbit IgG, Promega) was performed on whole-mount preparations of the finely dissected organ of Corti. We incubated the tissues in the antibody solutions for 1 h after blocking. As secondary antibodies, we used Cy3–conjugated anti rabbit IgG (Sigma Aldrich). F-actin was visualized with Alexa 633–conjugated Phalloidin (Life technologies). Fluorescence confocal images were obtained with a LSM510-META confocal microscope (Carl Zeiss). Some of the green fluorescence in Cx26R75W + mice indicated the pseudocolor obtained from the signal of Alexa 633–conjugated secondary antibodies (Invitrogen), because these mice have ubiquitous EGFP expression from their transgene. z-stacks of images were collected at 0.5 μm intervals, and the single image stacks were constructed with LSM Image Browser (Zeiss); three-dimensional images and videos were constructed with IMARIS software (Bitplane). We analyzed at least six samples from three animals, and representative images are shown. The compared images were photographed and processed using identical parameters. Three-dimensional images were constructed with z-stacked confocal images by IMARIS (Bitplane).

Neurospheres for neuronal differentiation, or HEK293T cells with transient overexpression were cultured on coverslips in 6-well plates. Afterward, cells were washed with phosphate buffered saline (PBS) thrice, fixed with 4% PFA for 15 min, which was inactivated with 50 mM ammonium chloride in PBS for 10 min. Cells were then permeabilized with 0.1% Triton X-100 for 10 min, blocked with 0.2% fish skin gelatin (Sigma-Aldrich, #G7041) for 30 min at room temperature. Subsequently, cells were incubated with primary antibodies against SOX1 (Abcam, #ab87775. 1:500), PAX3 (Abcam, # ab15717. 1:200), MAP2 (CST, #8707. 1:200), TUBB3 (CST, #5568. 1:200), HA-tag (CST, #2367. 1:500), Myc-tag (Sigma, #C3956. 1:500), nonP-β-CAT (CST, #8814. 1:500), LSD1 (CST, #2139. 1:500), SMAD2 (CST, #5678. 1:500), SMAD4 (CST, #9515. 1:500), DNMT1 (CST, #5032. 1:500), HDAC1 (CST, #5356) at 4°C overnight. The secondary antibody was Cy3-conjugated anti-rabbit IgG (Sigma-Aldrich, #C2306. 1:1,000), anti-mouse IgG (FITC-conjugated) (Sigma-Aldrich, #F9137. 1:1,000), and Alexa Fluor^®568 donkey anti-Rabbit IgG (H+L) (Invitrogen, #A10042. 1:1,000). Cells were counterstained with DAPI to view cell nuclei. After being rinsed, coverslips were mounted with anti-fade mounting medium (Invitrogen, #S36936). Cells were then detected using fluorescence microscope (FluoView FV1000, Olympus, Leica TCS SP5 II).

HEK293 cells were seeded (1 × 10⁵ cells/mL) on coverglass bottom dishes (SPL Life Sciences, Pocheon, South Korea), grown for 24 h, and then treated with various concentrations of HU for 1 h. Cells were fixed with 4 % paraformaldehyde in PBS for 20 min and permeabilized with 0.1 % Triton X-100 in PBS for 10 min. Blocking was performed with 15 % FBS in PBS for 15 min at 37 °C. Cells were incubated with the indicated primary antibodies at 37 °C for 30 min, washed three times with PBS, and then incubated with Alexa Fluor^® 488-conjugated anti-mouse IgG (Invitrogen) and Cy3-conjugated anti-rabbit IgG (Sigma-Aldrich) for 30 min at 37 °C. Cells were washed three times with PBS, and the nucleus was counterstained with To-Pro^®-3 (Invitrogen). Finally, the cells were analyzed using a confocal fluorescence microscope (Olympus FV-1000; software, Olympus FluoView; Olympus, Center Valley, PA, USA).