Alexa fluor 594 anti rabbit igg

Rabbit monoclonal antibody (MAb) C29F4 against the HA epitope was purchased from Cell Signaling Technology (Leiden, The Netherlands). Mouse MAbs against HEV ORF2 (1E6), MAVS (Adri-1), GM130 (clone 35), and β-actin (AC-15) were from Millipore (Burlington, MA), AdipoGen (San Diego, CA), BD Biosciences (San Jose, CA), and Sigma-Aldrich (St. Louis, MO), respectively. Mouse MAbs against ERGIC-53 (OTI108), PDI (1D3), and CLIMP63 (G1/296) were from Enzo Life Sciences (Farmingdale, NY), and MAbs against CD63 (MX491295) and CD151 (11G5a) were from Santa Cruz Biotechnology (Dallas, TX). Recombinant mouse antibody MRB198 against genotype 3 HEV ORF3 protein have been described previously (11 (link)). Alexa Fluor 488 and Alexa Fluor 694 anti-mouse IgG as well as Alexa Fluor 594 anti-rabbit IgG secondary antibodies were from Thermo Fisher Scientific. Horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG secondary antibodies were from GE Healthcare (Chicago, IL) and Agilent Technologies (Santa Clara, CA), respectively.

Snails were fixed in Halmi’s fixative (4.5% mercuric chloride, 0.5% sodium chloride, 2% trichloroacetic acid, 20% formol, 4% acetic acid, and 10% picric acid-saturated aqueous solution). Embedding in paraffin and transverse histological sections (10 µm) were performed. The slides were stained using Anti-BgTEP-PEP antibody according to a previously developed protocol (36 (link)).
Slides re-hydration was performed in serial toluene, 95, 70, and 30% ethanol and finally PBS bathing (saline phosphate buffer: pH 7.4–7.5; 8.41 mM Na₂HPO₄; 1.65 mM Na₂H₂PO₄; 45.34 mM NaCl; H₂O milliQ q.s.p.). The slides were immersed in a permeabilizing PBS solution containing 0.5% Triton X-100 during 15 min. A saturation step was performed in PBS buffer containing 1% gelatin hydrolyzate (Bellon, France), 1% normal goat serum (NGS, Sigma), and 0.1% NaN₃ (Sigma) during 1 h at room temperature. Slides were then successively incubated with the anti-BgTEP-PEP antibody dilution 1:100 for 1 h 30 min at room temperature and with an Alexa Fluor 594 anti-rabbit IgG (Thermo Fisher Scientific) diluted 1:1,000 for 1 h at room temperature. Slides were mounted in Vectashield and stored in dark at 4°C. The slide observation was carried out by epifluorescence and light microscopy using a Zeiss axioscope 2 microscope (Carl Zeiss AG) and a Leica DC350FX camera (Leica).

The primary antibodies used in this study were as follows: anti-RECQL4 antibody was prepared as previously described (24 ). Other antibodies used in this study included anti-pATM (Ser-1981, #4526, Cell Signaling), anti-MRE11 (GTX30294, GeneTex), anti-NBS1 (A7703, ABclonal), anti-RAD51 (GTX100469, GeneTex), anti-USP2 (A10399, ABclonal), anti-USP28 (A9292, ABclonal), anti-γH2AX (A300-081A, Bethyl and 05-636, EMD Millipore), anti-Lamin B1 (ab16048, Abcam), anti-GFP (sc-9996, Santa Cruz), anti-FLAG M2 (Sigma), and anti-HA (AE008, ABclonal). Alexa Fluor 488 antimouse IgG (A11001, ThermoFisher), Alexa Fluor 488 anti-rabbit IgG (A11008, ThermoFisher), Alexa Fluor 594 antimouse IgG (A11005, ThermoFisher), and Alexa Fluor 594 anti-rabbit IgG (A11012, ThermoFisher) were used as secondary antibodies for immunofluorescence microscopy.

Mice were deeply anesthetized, transcardially perfused, and fixed with 10 mL ice-cold phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA). The whole brains were dissected out, further fixed in 4% PFA for 60 min, and soaked in 15% sucrose solution until completely sinking at 4°C. The cerebellums were removed from the whole brains, and cerebra were frozen in dry ice and then in a -80°C deep freezer (Sanyo, Tokyo, Japan). Specimens containing the striatum area were cut into 10-μm thick sections using a Cryostat (Leica, Wetzlar, Germany) and placed on CREST-coated slide glasses (Matsunami Glass, Osaka, Japan). After rinsing with PBS, the sections were blocked and permeabilized with 5% goat serum/0.5% Triton X-100 in PBS for 60 min at room temperature. Then, the sections were incubated with mouse anti-IDO (1:200) (Chemicon) and rabbit anti-tryptophan hydroxylase (TPH)2 (1:200) (Cell Signaling Technology, Danvers, MA, USA) antibodies overnight at 4°C. After rinsing with PBS, the sections were incubated with corresponding Alexa Fluor 488 anti-mouse IgG (1:250) and Alexa Fluor 594 anti-rabbit IgG (1:250) (Thermo Fisher Scientific) under dark conditions and room temperature for 60 min. The fluorescence signals from these sections were acquired using a confocal fluorescence microscope (Leica).

D52-2-7 cells were treated with 2 µg/ml (7.1 µM) BFA, or vehicle only (0.02% (v/v) DMSO) for 5 h at 37 °C, fixed in 3% (w/v) formaldehyde in PBS for 15 min at RT, then in cold acetone/methanol (v/v, 1:1) for 15 min at −20 °C. Cells were then stained by TPD52 rabbit monoclonal Ab (1:100) and Adipophilin/PLIN2 guinea pig polyclonal antibody (1:100) diluted in 3% (w/v) BSA/PBS overnight at 4 °C. Alexa Fluor®594 anti-rabbit IgG (2 µg/mL, Thermo Fisher Scientific, VIC, AU) and Alexa Fluor^®633 anti- guinea pig IgG (2 µg/mL, Thermo Fisher Scientific, VIC, AU) were utilised as secondary antibodies at 1:200 dilution, and coverslips were mounted onto slides using Prolong Diamond Antifade Mountant (Thermo Fisher Scientific, VIC, AU). STED microscopy^{84 (link)} was performed on a Leica TCS SP8 STED 3X microsystem, located in Australian Microscopy & Microanalysis Research Facility at the Australian Centre for Microscopy & Microanalysis at the University of Sydney. The system was equipped with a pulsed white light excitation laser (470–670 nm), and 592 nm and 775 nm lasers for fluorophore depletion by STED. Images were taken using a HC PL APO 100×/1.4 oil STED WHITE objective lens. Imaging and setup of scans, including STED alignment, were performed using LAS AF software (Leica, NSW, AU).