Alexa fluor 488 conjugated goat anti mouse igg secondary antibody

NPCs were fixed in 4% paraformaldehyde, washed in three changes of PBS and then incubated overnight with a monoclonal antibody directed against cytochrome c (BD PharMingen, San Diego, CA, USA). Cells were then washed and incubated for 1 h with AlexaFluor 488-conjugated goat anti-mouse IgG secondary antibody (Invitrogen) and counterstained with Hoechst 33258 (1 μg/ml). To evaluate mitochondrial membrane permeabilization, cells were visualized by fluorescence microscopy and cells exhibiting punctate, cytoplasmic cytochrome c staining were considered to have maintained membrane integrity, whereas cells lacking cytoplasmic cytochrome c staining were considered to have undergone mitochondrial membrane permeabilization as we have described previously.^{67 (link)} Images were captured and scored by a blinded observer and a minimum of 400 cells were analyzed per well.

Formononetin was obtained from Yick-Vic Chemicals & Pharmaceuticals Ltd, (Hong Kong). Antibodies for GSK-3β and adenine nucleotide translocase (ANT) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against phospho-GSK-3β (Ser9), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), Akt, and phospho-Akt were obtained from Cell Signaling Technology (Boston, MA, USA). Anticyclophilin D (anti-Cyp-D) was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Alexa Fluor 594-conjugated goat anti-rabbit IgG secondary antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody were obtained from Invitrogen (Carlsbad, CA, USA).

Yeast cells expressing wDIII were stained with mouse sera or the E16 mAb as described previously [24 (link)]. Briefly, yeast cells were grown to log phase and induced for wDIII expression. Pooled mice sera collected in week 11 of the HBcAg-wDIII VLP immunization experiments were diluted with PBS (1:1000) and then incubated with yeast cells. Pooled sera from the PBS mock-immunized mice was used as a negative control. The specific binding of antibodies in immunized mouse sera to wDIII on yeast cells was detected by staining yeast with an Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody (Invitrogen, Waltham, MA, USA). For a competitive binding assay, yeast cells were pre-incubated with mouse sera and then incubated with E16, a potent neutralizing humanized IgG1 mAb against wDIII. After washing, yeast cells were stained with an Alexa Fluor 488-conjugated goat anti-human IgG secondary antibody (Invitrogen, Waltham, MA, USA). Yeast cells stained with Alexa Fluor 488-conjugated secondary antibodies were then analyzed on a BD FACSCalibur flow cytometer.

BMMs were cultured overnight at a density of 5 × 10³ cells/well in 96-well plates with complete α-MEM and 50 ng/ml M-CSF. Subsequently, BMMs stimulation was done using 50 ng/ml RANKL for mature osteoclasts formation in the presence or absence of various DHD concentrations (1 and 2 μM). Then, cells were fixed in 4% paraformaldehyde (PFA) for 10 min and permeabilized for 5 min with 0.1% Triton X-100. After that, cells were blocked in phosphate-buffered saline (PBS) containing 3% bovine serum albumin (BSA) for half an hour and then underwent incubation in the darkroom for 45 min for F-actin ring staining with rhodamine-conjugated phalloidin. BMMs were incubated overnight at 4°C with Vinculin Recombinant mouse monoclonal primary antibody (Invitrogen, Rockford, Illinois, United States) and then labeled at room temperature for 45 min with Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody (Invitrogen, Rockford, Illinois, United States). After being rinsed with PBS and counterstained with DAPI (Santa Cruz, United States), images were captured using a confocal fluorescence microscope (Nikon, A1 PLUS, Tokyo, Japan).

For the detection of poly(A)⁺ RNA in T. brucei and T. cruzi, the parasites were harvested, washed in PBS (pH 7.4), fixed by incubation in 4% paraformaldehyde and were allowed to adhere to poly-L-lysine-coated slides for 10 minutes. The slides were washed in PBS and the parasites were permeabilized by incubation with 0.2 M HCl (diluted in PBS) for 10 minutes. For T. cruzi, the cells were incubated with prehybridization buffer (35% formamide, 0.02% BSA in 2X SSC buffer) supplemented with 25 µg/ml tRNA, 1 mg/ml salmon sperm DNA (Sigma-Aldrich) and 40 U/ml RNaseOUT (Invitrogen) for 30 minutes at 37°C. For T. brucei, the cells were incubated with prehybridization buffer containing 10X Denhardt’s solution, 1 mM EDTA, 35% formamide in 4X SSC and supplemented with 0.5 µg/ml tRNA and 2 mU/ml RNaseOUT for 30 min at room temperature. Digoxigenin-conjugated oligo(dT) probes (6 ng/µl) were diluted in prehybridization buffer and denatured by heating at 65°C for 3 minutes. Hybridization was performed for 16 hours at 37°C. Probe binding was detected by indirect immunofluorescence analysis with mouse monoclonal anti-digoxigenin antibody (Sigma-Aldrich, 1:300 dilution) and Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody (Invitrogen, 1∶600 dilution), as described previously. As a control, 100 µg/ml RNase A was added to the pretreatment buffer before probe hybridization.