Alexa fluor 647 donkey anti goat igg h l

Specific antibodies for Flag (M3165), α‐tubulin (T6199), and β‐actin (A2228) were acquired from Sigma (MO, USA). Those antibodies for Olfm4 (39141), β‐TrCP (4394), and HA (3724) were acquired from Cell Signaling Technology (MA, USA); those for Ki67 (ab15580), Lgr5 (ab75732), and lysosome (ab2408) were from Abcam (Cambridge, UK); those for β‐catenin (610153) were from BD Biosciences (San Jose, CA); those for SOX9 (AB5535) were from EMD Millipore (MA, USA); those for GSK3α/β (sc‐7291) and Laminin A/C (sc‐7292) were from Santa Cruz (Santa Cruz, CA); and those for MST4 and phospho‐β‐catenin (Thr40) were produced by Shanghai Immune Biotech (Shanghai, China). Secondary antibodies for Alexa Fluor 568 goat antirabbit IgG (H+L) (A11011), Alexa Fluor 647 donkey antigoat IgG (H+L) (A21447), and Alexa Fluor 488 goat antirabbit IgG (H+L) (A11001) were acquired from Invitrogen (Carlsbad, CA).

Femurs with drill hole injury were dissected free of connective tissues, fixed in 4% buffered formalin, decalcified in 1% EDTA and embedded in paraffin. Transverse sections of 5μm were then cut from each sample. Sections were initially deparaffinized using xylene, rehydrated through an ethanol gradient and permeabilized with 0.1% Triton X-100 followed by blocking with 1% BSA. These were then incubated with β-catenin antibody diluted in 0.5% BSA (1:100) at 4°C overnight. After washings in 1× PBS, sections were incubated with Alexaflour 488 goat anti-rabbit (1:500). Sections were then washed with PBS and incubated with ALP antibody (1:100) diluted in PBS containing 0.5% BSA, overnight at 4°C under humid conditions. Sections were again washed with PBS and incubated with fluorescent Alexa Fluor-647 donkey anti-goat IgG (H + L) (1:500 dilution in PBS) (Molecular Probes, Carlsbad, CA,USA) at room temperature for 1 hour. Sections were counter stained with DAPI for 15 minutes in dark. After 15 min incubation slides were rinsed with PBS and mounted with antifade mounting media ((life technologies, Carlsbad, CA, USA). Sections were visualized under Cell Imaging Station (life technologies, Carlsbad, CA, USA).

EP and 8-Br-cAMP were purchased from Sigma-Aldrich (St. Louis, MO, USA). SB203580 and H89 were purchased from Cayman Chemical Company (Ann Arbor, MI, USA) and EMD Millipore (Temecula, CA, USA), respectively. The following antibodies were used in this study: primary antibodies: anti-mouse FLAG M2 monoclonal (F1804; Sigma-Aldrich), anti-mouse γH2AX monoclonal (05-636; EMD Millipore), anti-rabbit p38MAPK polyclonal (#9212; CST, Danvers, MA, USA), anti-rabbit GADD45A polyclonal (sc-792; Santa Cruz Biotechnology, Dallas, TX, USA), anti-rabbit phospho-p38MAPK (Thr180–Tyr182) polyclonal (#9211; CST), and anti-goat CYP21A2 polyclonal (C-17) (sc-48466, Santa Cruz, Santa Cruz, CA, USA); and secondary antibodies: Alexa Fluor 647 goat anti-mouse/rabbit IgG (H + L) (A-21236/A-21245; Molecular Probes, Eugene, OR, USA), Alexa Fluor 647 donkey anti-goat IgG (H + L) (A-21447; Molecular Probes), Alexa Fluor 546 goat anti-mouse IgG (H + L) (A-11030, Molecular Probes), and Alexa Fluor 546 donkey anti-mouse/rabbit IgG (A-10036/A-10040; Molecular Probes).

Frozen sections 6–8 μm thick were fixed in ice‐cold acetone, rehydrated in PBS and blocked with normal horse serum before antibody application. The following antibodies were purchased from BioLegend: anti‐CD1d (1B1), anti‐CD4 (RM4‐5), anti‐CD21/35 (7E9) and anti‐CD45R/B220 (RA3‐6B2). The following antibodies were purchased from BD Biosciences: anti‐CD35 (8C12), anti‐MAdCAM‐1 (MECA‐367) and anti‐TNP (G235‐1). Anti‐CD169 (MOMA‐1) and anti‐MARCO (ED31) were purchased from Bio‐Rad (Hemel Hempstead, UK). Anti‐CD209b/SIGNR1 (eBio22D1) and phycoerythrin (PE)‐conjugated anti‐Armenian hamster IgG were purchased from eBiosciences (ThermoFischer, Loughborough, UK). Anti‐CXCL13 (polyclonal) was purchased from R&D Systems. Streptavidin Alexa Fluor 594, goat anti‐rat IgG (H+L) Alexa Fluor 594, donkey anti‐goat IgG (H+L) Alexa Fluor 647 and goat anti‐rat IgG (H+L) Alexa Fluor 488 were purchased from ThermoFisher Scientific (Waltham, MA). Dako fluorescent mounting medium (Agilent, Santa Clara, CA) was used to apply coverslips before image acquisition. A Zeiss LSM5 Pascal (Carl Zeiss, Oberkochen, Germany) upright microscope with zen software (Rochdale, UK) was used for image collection.

For immunofluorescence, primary TGN46 antibody (Cat. No. AHP500GT, Bio-Rad Laboratories, Hercules, CA, USA) was used at a concentration of 1:250. Goat anti-GRASP65 antibody (Cat. No. sc-19481, Santa Cruz Biotechnology, Dallas, TX, USA) and goat anti-GM130 antibody (Cat. No. sc-16268, Santa Cruz Biotechnology, Dallas, TX, USA) were used at a concentration of 1:50. Sheep-488 (Cat. No. A-11015, Thermo Fisher Scientific, Waltham, MA, USA) and Donkey Anti-Goat IgG H&L Alexa Fluor 647 (Cat. No. ab150131, abcam, Cambridge, UK) were used as a secondary antibody at a concentration of 1:200 and 1:250, respectively.