Goat anti rabbit igg conjugated to alexa fluor 488

For live cell imaging studies, 1 × 10⁵ U251 cells were seeded in 35-mm dishes and incubated for 24 h before commencing treatment with compounds. Cells were examined by phase-contrast microscopy using an Olympus IX70 inverted microscope equipped with a heated stage, a DP80 digital camera, and cellSens™ software (Olympus America, Center Valley, PA).
For immunofluorescence localization of Glut1, cells were grown on coverslips, treated with MOMIPP or DMSO for 4 h, washed with PBS, and then fixed in ice-cold methanol. After pre-blocking with 10% goat serum, cells were incubated with anti-Glut1 polyclonal antibody (Millipore, Cat. No. 07–1401) at 1/50 dilution for 1 h, followed by goat-anti-rabbit IgG, conjugated to AlexaFluor-488 (ThermoFisher) at 1/600 dilution. Fluorescence images were captured with an Olympus IX70 inverted fluorescence microscope, using a DP80 digital camera, and cellSens™ software (Olympus America).

Primary hippocampus neuron cultures were prepared from embryonic days 16 to 18 (E16–18) in C57BL/6 mouse brains according to the previous study. All experimental procedures were performed according to the guidelines of the animal research committee of Zhujiang Hospital, Southern Medical University. Dissociated hippocampal neurons were plated onto poly-D-lysine-coated confocal dish at a density of 500,000 cells/dish. For each dish, 20 μL of 0.1 mg/ml of sufficiently sonicated PFFs will be added. The working concentration of the human or mouse PFFs was 1 ug/ml. Treated neurons were harvested for immunocytochemistry at 10 to 15 days post-PFFs treatment (17–22 DIV). To determine if the LB/LN pathologic aggregates were triggered by exogenous PFFs, the treated mouse primary hippocampus neurons were fixed with 4% paraformaldehyde/4% sucrose in PBS, followed by permeabilization with 0.1% Triton X-100. Then, neurons were stained with the monoclonal rabbit primary antibody against α-synuclein phosphorylated at S129 (1:1000; Abcam, ab51253, United States) overnight at 4°C and incubated with goat anti-rabbit IgG conjugated to Alexa Fluor 488 (1:2000, Thermo Fisher Scientific, Waltham, MA, United States) for 1 h at RT. Fluorescence images were captured by Zeiss LSM 880 (Carl Zeiss, Jena, Germany) laser-scanning microscope and analyzed by using a ZEN lite 2012 software.

Cells were cultured in LB medium containing ampicillin and 500 µM IPTG (no IPTG was added to puppS baseline [BL]) at 37 °C overnight. Total cell material was matched by centrifuging the equivalent of 1 ml of culture at an OD₆₀₀ = 1.0 and resuspending the pellet in 20 µl of PBS. 3 µl aliquots were then applied to a nitrocellulose membrane and allowed to dry for 20 min. Membranes were blocked with 5% skimmed milk in PBS for 1 h. Rabbit anti-ECA serum (Statens Serum Institut) was then applied at 1:1,000 in PBS for 3 h. The primary antibody was detected by using the goat anti-rabbit IgG conjugated to Alexa Fluor 488 (Thermo Fisher Scientific) secondary antibody and was applied at 1:4,000 for 1 h. Blots were imaged by using a ChemiDoc MP imaging system (Bio-Rad Laboratories). Signals corresponding to surface ECA were quantified by using ImageJ.

Phenylephrine (Phe, α receptor agonist), acetylcholine, endothelin 1 (ET-1, endothelin receptor agonist), adenosine 5′-triphosphate disodium salt (ATP-Na₂), 2-aminoethyl diphenylborinate (2APB, inositol triphosphate receptor inhibitor) were purchased from Sigma and dissolved in the distilled water. Thapsigargin (TG) was obtained from Calbiochem and dissolved in dimethyl sulfoxide (DMSO). The primary goat (sc-10,377) and rabbit (sc-25,749) antibodies against TRPP2, the primary rabbit antibody against IP₃ receptor and the primary rabbit antibody against Orai1 were purchased from Santa Cruz Biotechnology. The primary rabbit antibody against STIM1 was obtained from ProSci. Fluo-4 fluorescence dye, TRPP2 small interfering RNA (siRNA), STIM1 siRNA, RNAiMax reagent, lipofectamine 2000, goat anti-rabbit IgG conjugated to Alexa Fluor 488 were purchased from Invitrogen. Protein A magnetic bead was obtained from Millipore.

Cells were grown at 37°C on coverslips in 12-well tissue culture plates in a culture medium containing 10% FCS (Life Technologies), antibiotics, and antimycotics. After reaching 70% confluence, the cells were washed with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde for 10 minutes, washed three times with PBS, and permeabilized with 0.25% Triton X-100 (PBST) at room temperature for 30 minutes. Next, cells were washed with PBS, blocked with 1% bovine serum albumin (BSA, Sigma-Aldrich) in PBST, and incubated with primary antibodies at 4°C (primary antibodies were diluted with 1% BSA PBST) overnight. After three washes with PBS, the cells were incubated in the dark with the corresponding fluorescein-conjugated secondary antibodies for 1 hour. The secondary antibodies used were goat anti-rabbit IgG conjugated to Alexa Fluor 488 (Invitrogen, catalog no. A11008), goat anti-mouse IgG conjugated to Alexa Fluor 488 (Invitrogen, catalog no. A11029), and donkey anti-mouse IgG conjugated to Alexa Fluor 594 (Invitrogen, catalog no. A21203). Subsequently, the cells were washed and mounted on slides. The nuclei were stained with 4’,6’-diamidino-2-phenylindole, and dihydrochloride (DAPI) using VECTASHIELD reagent (Vector Laboratories) and visualized using an LSM 710 confocal laser scanning microscope (Carl Zeiss) equipped with ZEN 2008 software (Carl Zeiss).

Whole eyes from 10- to 12-week-old mice were fixed in 4% PFA in PBS, pH 7.4, overnight at 4 °C, cryoprotected in 30% sucrose in PBS, pH 7.4, frozen in optimal cutting temperature medium, and cryosectioned into 10 μm-thick sections. After washing away the medium, the sections were treated using heat-mediated antigen retrieval by boiling in 10 mM sodium citrate, pH 6.0, with 0.05% Tween-20 for 1 h. After cooling, the slides were washed in PBS and subsequently blocked in 10% goat serum/5% BSA/0.5% Triton X-100 in PBS for 1 h at RT. Following blocking, the sections were incubated with primary antibodies against peptidyl arginine deiminase 4 (PAD4, ProteinTech, Rosemont, IL, USA) and citrullinated peptides (Clone F95, EMD Millipore, Burlington, MA, USA) O/N at 4 °C. After washing in PBS, the slides were probed using goat anti-rabbit IgG conjugated to AlexaFluor488 (Invitrogen) and goat anti-mouse IgM conjugated to AlexaFluor555 (Invitrogen), and the nuclei were labeled with DAPI (Invitrogen). After washing in PBS, the slides were mounted using Prolong Diamond Anti-Fade Mountant (Invitrogen) and imaged using a Zeiss 710 Laser Scanning Confocal Microscope (Zeiss, Oberkochen, Germany). All images were captured as Z-stacks and expressed as maximum intensity projections.

MDBK monolayers infected for 4 h with E. tenella sporozoites were fixed in 4% paraformaldehyde in PBS for 15 min and washed in PBS. Fixed monolayers were blocked by incubating in 3% BSA, 0.25% Triton ×100 in PBS for 30 min then in rabbit anti-EtMIC2 serum (1/500) for 1 h. After three washes in PBS, monolayers were incubated with goat anti-rabbit IgG conjugated to Alexa Fluor 488 (1/1000; Invitrogen), washed in PBS, then observed under UV fluorescence using a Leica DMI3000B microscope and photographed with a Leica DCF365FX camera. Image processing was performed using the LAS AF (Leica Microsystems).