Alexa 594 goat anti mouse igg

Specific antibodies were purchased from the following commercial sources: Anti-AFP, anti-ALB, anti-CD44, anti-Evi1, anti-flag (mouse), anti-HA, anti-HNF4α, anti-H3, anti-H3K36me3, anti-Myc, anti-OCT4A, anti-P300, anti-PPM1A (rabbit), anti-pSmad2 (S465/467), anti-pSmad2 (S245/250/255), anti-pSmad3 (s423/425), anti-Smad2, anti-Smad3 (rabbit), anti-SnoN, anti-Sox2, anti-TAT, and normal rabbit IgG from Cell Signaling Technology (Danvers, MA); anti-PPM1A (mouse), anti-SC35, and anti-SETD2 were from Abcam (Cambridge, MA); Anti-Smad4 and normal mouse IgG were from Santa Cruz Biotechnology (Santa Cruz, CA); Anti-β-actin, and anti-flag (rabbit) from Sigma-Aldrich (St. Louis, MO); Alexa594 goat anti-mouse IgG from Life Technology (Carlsbad, CA); Dylight488 goat anti-rabbit IgG from Vector Labs (Burlingame, CA).

Cells were washed with PBS, then fixed with 4% paraformaldehyde (P6148, Sigma-Aldrich, St. Louis, MO, USA) for 30 min at room temperature. After two washes with PBS, cells were incubated with a blocking buffer (10% goat serum and 0.3% Triton X-100 in PBS) for 1 h. Primary antibodies were diluted in blocking buffer and then added to cells for 2 h at room temperature and then incubated with antibodies against Math5 (1:500; Millipore, Bethesda, MA, USA), Brn3b (1:250; Santa Cruz, Dallas, TX, USA), and Tuj1 (1:500; Abcam, Cambridge, UK). The cells were then subjected to three 3-minute washes with PBS and incubated with secondary antibodies diluted in a blocking buffer containing Hochest (1:10,000; Thermo Scientific, Waltham, MA, USA) for 1 h at room temperature. The following secondary antibodies were used: Alexa 488 goat anti-rabbit IgG, Alexa 594 goat anti-rabbit IgG, Alexa 488 goat anti-mouse IgG, and Alexa 594 goat anti-mouse IgG (Thermo Scientific, Waltham, MA, USA). After three 3-minute washes with PBS, the cells were visualized using confocal microscopy (Zeiss LSM 700, Zeiss, Jena, Germany).

Live cells were cultured in MitoTracker Red (Cell Signaling, Danvers, MA, USA) at 37 °C for 30 min and then with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 30 min. Next, cells were incubated with blocking buffer containing 10% goat serum and 0.3% Triton X-100 in PBS for 1 h and then incubated with primary antibodies against Brn3b (1:250; Santa Cruz, Dallas, TX, USA), Math5 (1:500; Millipore, Bethesda, MA, USA), and Thy1 (1:500; Abcam, Cambridge, UK) at 4 °C overnight. The cells were then washed with PBS and incubated with secondary antibodies diluted in a blocking buffer containing Hoechst (1:10,000; Thermo Scientific, Waltham, MA, USA) for 1 h at room temperature. The following secondary antibodies include sheep and Alexa 488 goat anti-rabbit IgG, Alexa 594 goat anti-rabbit IgG, Alexa 488 goat anti-mouse IgG (Thermo Scientific, Waltham, MA, USA), and Alexa 594 goat anti-mouse IgG (Thermo Scientific, Waltham, MA, USA). Fluorescent staining of mitochondria is then performed using the MitoTracker Red CMXRos (Cell Signaling, Danvers, MA, USA). Finally, the cells were detected by confocal microscopy (Zeiss LSM 700, Zeiss, Jena, Germany).

The following antibodies were used: mouse anti-VP5 (Virusys, HA018; 1:300); mouse anti-ICP8 11E2 (Abcam, #20194; 1:100); mouse anti-ICP4 (Virusys, H1A021; 1:400); mouse anti-α-tubulin (Sigma-Aldrich, #T6074; 1:1000); rabbit anti-PML [73 (link)] (1:300); mouse anti-cyclin B1 (Abcam, ab18221; 1:500); Alexa-594 Goat anti-mouse IgG (Thermofisher; 1:750).

36-48 hours after COS7 cells transfection, the transfected cells were washed twice with PBS, fixed with 4% paraformaldehyde, and washed three times with PBS. Cells were then permeabilized and blocked with normal goat serum, Triton X-100, and NaN₃ in PBS for 1 hour at room temperature. ATP11A protein was labeled with mouse anti-Flag antibody (1 : 2000, Sigma, Germany), and the endoplasmic reticulum (ER) was labeled with a rabbit anti-Calnexin antibody (1 : 1000, Cell Signaling Technology, CA, USA). The Golgi apparatus was labeled with a rabbit anti-GM130 antibody (1 : 1000, BD Biosciences, Mississauga, ON). The secondary antibodies used were Alexa 488 goat anti-rabbit IgG, Alexa 594 goat anti-mouse IgG (1 : 500, Invitrogen, USA). The images were captured under a Zeiss LSM 800 confocal scanning microscope.

In this experiment, FITC-JM2 solution with a concentration of 150 µg mL⁻¹ was prepared by dissolving FITC-JM2 powder in RPMI-1640 or DMEM culture medium. B16F10 or 4T1 cells were seeded in glass bottom cell dish (Nest, 801002, China) at a density of 2 × 10⁴ cells per dish and cultured for 24 h. Then, the culture medium was replaced by 150 µg mL⁻¹ FITC-JM2 solution. After 4 h, the culture medium was discarded and the cells were washed twice with PBS. Then, the cells were fixed with 4% PFA for 10 min and permeabilized with 0.3% triton-x100 for 5 min. After that, the cells were blocked with 1% BSA-PBS solution at 37 °C. The cells were incubated with mouse anti-α-tubulin (Sigma, USA) primary antibody (diluted with 0.5% BSA-PBS solution at the ratio of 1:300) and kept at 4 °C overnight. The cells were then washed with PBS for three times and stained with Alexa 594 goat anti-mouse IgG (Invitrogen, USA) (diluted with 0.5% BSA-PBS at the ratio of 1:1000). At the end of the incubation, the cells were washed with PBS before the nuclei of the cells were stained with 5 µg mL⁻¹ DAPI solution. Finally, the stained cells were observed and photographed with a camera connected to a confocal laser scanning microscope. The PCC and the MOC were analyzed to quantify the degree of colocalization between JM2 and microtubules. Three independent experiments were carried out for validation.