Goat anti rabbit igg alexa 647

MC3T3-E1 cells were lifted from culture dish using Accutase (Biolegend) and incubated with WT or mutant CtsK at 100 ng/mL in PBS with 0.1% BSA for 1 hr at 4 °C. Bound CtsK was stained with our rabbit anti-mouse CtsK (1µg/mL) for 1 hr at 4 °C, followed by goat anti-rabbit IgG-Alexa 647 (1:1,000; ThermoFisher Scientific) for 30 min and analyzed by flow cytometry.
The rabbit anti-mouse CtsK antibody was developed by immunizing rabbit with a mixture of murine proCtsK and mature CtsK. The polyclonal antibody was affinity purified from the anti-serum with a column immobilized with proCtsK, and the purified antibody displayed strong binding to both proCtsK and mature CtsK.

Cells were rinsed twice with ice-cold PBS and mounted on slide glass using SmearGell (GenoStaff). The embedded samples were fixed with 4% paraformaldehyde/TBS for 60 min at room temperature. They were then rinsed twice with TBS, and permeabilized with 0.5% Triton X-100 in TBS for 60 min at room temperature. After rinsing twice with TBS, the samples were blocked for 60 min with 10% normal goat serum (Thermo Fisher Scientific), followed by rinsing twice with TBS. Incubate with anti-Ki-67 mouse monoclonal antibody (× 200) and anti-E-cadherin (24E10) antibody (× 200) for 60 min at room temperature. After rinsing twice with TBS, samples were incubated with goat anti-rabbit IgG-Alexa647 and goat anti-mouse IgG-Alexa488 (Thermo Fisher Scientific) (× 500) for 60 min at room temperature. After washing with TBS twice at room temperature, slide glass was mounted with Prolong Gold Antifade mountant with DAPI (Thermo Fisher Scientific). Images were acquired using BZ-X800 (Keyence). All the images were obtained and analyzed in a single setting. The primary antibodies used were Ki-67 (8D5) (Cell Signaling Technology #9449S) and E-cadherin (24E10) (Cell Signaling Technology #3195S).

MC3T3-E1 cells were lifted from culture dish using Accutase (Biolegend) and incubated with WT or mutant hOPG at different concentrations (30–5000 ng/mL) in PBS with 0.1% BSA for 1 h at 4 °C. Bound hOPG was stained with our rabbit anti-mouse/human OPG (1 µg/mL) for 1 h at 4 °C, followed by goat anti-rabbit IgG-Alexa 647 (1:1,000; ThermoFisher Scientific) for 30 min and analyzed by flow cytometry. In some experiments, cells were pretreated with recombinant heparin lyases III (5 milliunits/ml, produced in house as a recombinant protein in E. coli) for 15 min at room temperature prior to binding experiments.
The rabbit anti-mouse/human OPG antibody was developed by immunizing rabbit with full-length murine OPG, and it shows strong cross-reactivity with human OPG. The antibody was affinity purified from the anti-serum with a column immobilized with recombinant murine OPG.

JAK3, STAT5, phospho-STAT5, phospho-I3K, phospho-ERK1/2, mitogen-activated protein kinase (MAPK), cyclin D, and cyclin E, cells were first stained for cell surface markers after which the cells were fixed for 10 min at 37 °C using 2% paraformaldehyde, and permeabilized by incubating the cells in 90% methanol for 30 min on ice. Subsequently, the cells were stained with anti-JAK3, anti-STAT5, anti-phospho-STAT5, anti-phospho-Akt, anti-phospho-ERK1/2, anti-cyclin D, or anti-cyclin E. As secondary antibody, goat-anti-rabbit IgG Alexa 647 (A21245, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) or goat-anti-mouse IgG Alexa 488 (A11029, Invitrogen, Thermo Fisher Scientific, Waltham, MA USA) was used. For the detection of phospho-STAT5, cells were stimulated with medium or 100 U/mL IL-2 for one hour at 37 °C prior to fixation. Cells were measured on a CyAn^TM ADP Analyzer equipped with a 488, 561, and 630 nm laser (Beckman Coulter, Brea, CA, USA), and analyzed with Summit 4.3.

The half-brains were fixed overnight, equilibrated in 30% sucrose in PB overnight, frozen in cold isopentane, and stored at −80 °C. The frozen brains were then cryosectioned into 30 μm sections on a −25 °C freezing stage microtome and free-floating sections were stored in a cryoprotectant solution until assayed. For immunofluorescence protocols, sections were washed to remove the cryoprotectant, blocked with 10% normal goat serum, and incubated in primary antibody for 48 h at 4 °C. For Aβ immunostaining, sections were incubated with 70% formic acid for 3 min after the first wash and washed prior to the blocking step. After primary antibody incubation, the sections were washed and incubated with secondary antibodies bound to Alexa fluorophores (Invitrogen) for 2 h at room temperature. Sections were mounted and cover slipped with Prolong Gold (Thermo Fisher, Waltham, MA, USA). Primary and secondary antibody dilutions were as follows: biotinylated 6E10 (BioLegend #803008) 1:2000; rabbit anti-Iba1 (Wako #019-19741) 1:3000; rat anti-LAMP1 (eBioscience #14-1071-82) 1:2000; rat anti-mDectin-1 (CLEC7A, InvivoGen #mabg-mdect, San Diego, CA, USA) 1:800; goat anti-rat IgG Alexa 488 (Invitrogen #A-11006) 1:1000; goat anti-rabbit IgG Alexa 647 (Invitrogen #A-21245) 1:1500, and Streptavidin Alexa 594 or 488 (Invitrogen #S11227) 1:1000.

The caudal half was prepared as described previously [28 (link)]. The head was incised at the median line to prepare two sagittal blocks before immunostaining. Whole-mount immunostaining was performed as described previously [28 (link)]. Primary antibodies to c-Kit (2B8, BD Biosciences or polyclonal goat IgG, R&D), CD45 (30-F11, BD Biosciences), and biotinylated anti-CD31 (MEC 13.3, BD Biosciences) were used in this study. The secondary antibodies (or streptavidin conjugates) used in this study were goat anti-rat IgG-Alexa647 (Invitrogen), Cy3-streptavidin (Jackson ImmunoResearch), Alexa488-streptavidin (Invitrogen), goat anti-rat IgG-Alexa555 (Invitrogen) and donkey anti-goat IgG-Dylight649 (Jackson ImmunoResearch). GFP and YFP were detected with rabbit anti-GFP antibodies (MBL), followed by goat anti-rabbit IgG-Alexa647 (Invitrogen).