Alexa 555 conjugated goat anti rabbit igg

The following antibodies were used: CD73 (BD Pharmingen 551123), CD105 (Sangon DA1348), Ep-CAM (HUABIO M1111-1), CD133 (HUABIO 0804-5), PAR (HUABIO 0902-5), BMP2 (HUABIO 0806-2), LGR5 (HUABIO R1107-8), IL1RL1 (HUABIO 0902-2), CD146 (HUABIO 0804-6), CD271 (HUABIO 1003-3), CgA (HUABIO M0407-29), Noggin (HUABIO 0805-2), Alexa 488-conjugated goat anti-rabbit IgG (Invitrogen), and Alexa 555-conjugated goat anti-rabbit IgG (Invitrogen).

Confocal immunofluorescence labeling of HCT116 cells was performed as described [28 (link)]. Briefly, cells were washed twice with cold PBS, fixed in 4% paraformaldehyde in PBS for 10 min at room temperature, permeabilized in 0.2% Triton X-100 in PBS for 5 min, and blocked in PBS containing 5% normal goat serum for 30 min at RT. Cells were then stained with primary antibody for overnight at 4°C. Following three washes, 10 min each, with PBS, the cells were incubated with Alexa 488-conjugated donkey anti-mouse IgG or Alexa 555-conjugated goat anti-rabbit IgG (Invitrogen) for 1 h at room temperature. After 3 × 10 min washes with PBS, cells were mounted with ProLong Gold Antifade Reagent (Invitrogen) and observed under a Zeiss LSM510 laser confocal microscope.
Statistical Analysis: Data are expressed as means ± standard error of the means (SEM). Statistical significance was determined by a 2-tailed unpaired Student’s t test or ANOVA. A p value of <0.05 was considered significant.

The samples were fixed in 4% paraformaldehyde and processed as per the standard procedure. Six-micron-thick sections were prepared for hematoxylin/eosin (H&E) staining and immunostaining. The specimens were boiled in citrate buffer (pH 6.0) for antigen retrieval and blocked using 1% goat serum or 5% bovine serum albumin in PBS. The specimens were incubated with primary antibodies against monoclonal rabbit anti-Ahnak (1:200, produced by Prof. Bae’s lab, Department of Life Sciences, Ewha Woman’s University, Seoul, Korea), monoclonal rabbit anti-p63 (1:200, GeneTex), monoclonal rabbit anti-upk1b (1:200, Invitrogen), monoclonal mouse anti-E-cadherin (1:200, BD Transduction), monoclonal rabbit anti-c-kit (1:100, Abcam), monoclonal mouse anti-α-SMA (1:200, Invitrogen) at 4 °C overnight. The following day, these sections were incubated with a secondary antibody Alexa488-conjugated goat-anti-rabbit IgG (1:200, Invitrogen), Alexa488-conjugated goat-anti-mouse IgG (1:200, Invitrogen), Alexa555-conjugated goat-anti-mouse IgG (1:200, Invitrogen), Alexa555-conjugated goat-anti-rabbit IgG (1:200, Invitrogen) and counterstained with TO-PROTM-3 (T3605, Invitrogen, OR, USA; 1:1000) to visualize nuclei. All specimens were examined using a confocal laser microscope (DMi8, Leica, Germany).

Immunofluorescence was performed as described in Rabelo et al., 2017 [25 (link)]. Antibodies were used at a dilution of 1:200 for a mouse monoclonal anti-Zika NS1 IgG (Arigo Biolaboratories, Taiwan, Republic of China), and a rabbit polyclonal antihuman c-Kit IgG (Santa Cruz, Texas, USA). After staining with primary antibodies, sections were incubated with an Alexa 488-conjugated rabbit anti-mouse IgG, Alexa 555-conjugated goat anti-rabbit IgG, or Alexa 555-conjugated goat anti-mouse IgG (ThermoFisher, Waltham, MA, USA). Slides were visualized by fluorescence microscopy (Olympus, Tokyo, Japan), and digital images were obtained using Image-Pro Plus software version 7.0.

Immunocytochemistry was performed as described previously^{21 (link)}. To visualize lipid droplets, cells grown on coverslips were fixed with 4% formaldehyde in PBS, and then BODIPY 493/503 (Thermo Fisher Scientific, Waltham, MA, USA) was stained for 30 min at room temperature. For immunofluorescent staining of cells, cells were treated as described in the figure legends. After stimulation, cells were fixed in 4% paraformaldehyde/PBS for 10 min and washed the cells with PBS. Cells were then permeabilized with 0.1% Triton X-100/PBS for 5 min and washed with PBS. Cells were blocked with 5% BSA/PBS for 1 h and then incubated with anti-LAMP-1 antibody in 5% BSA/PBS at 4 °C overnight. Cells were washed with PBS 3 times (5 min each) and then incubated with Alexa 555 conjugated-goat anti-rabbit IgG (Thermo Fisher, Waltham, MA, USA) for 1 h at room temperature. Images were acquired with an Axiovert fluorescence microscope (Carl Zeiss Ltd., Thornwood, NY, USA). Ten to fifteen cells were randomly selected from each treatment to calculate the average number of lipid droplets and the percentage of co-localization per cell. Quantification was performed using Image J software (NIH, MD, USA) and the percentage of co-localization was calculated by JACoP plugin of Image J. The data shown are from one representative experiment of three independent repeats.

MV3 cells were seeded on coverslips in 24-well plates (Starlab GmbH) overnight. After washing with DPBS, cells were fixed with 4% paraformaldehyde for 10 min at RT. Prior to staining, cells were permeabilized with 0.1% saponin for 15 min. Afterwards, cells were stained with TF antibody (rabbit anti-mouse and human, IgG 1:100, GeneTex GTX01033) for 1 h at RT. After washing with DPBS three times, cells were further stained with the secondary antibody: Alexa 555-conjugated goat anti-rabbit (IgG, Thermo Fisher Scientific, 1:500) for 45 min at RT. Nuclei were stained with DAPI for 20 min at RT. Finally, samples were imaged with a fluorescence microscopy (Observer z.1, Zeiss).