Alexa fluor 488 goat anti rabbit secondary antibody

HTR-8/SVneo cells were seeded in glass-bottom cell culture dishes and grown to 70% confluence. Then, the cells were fixed with 4% paraformaldehyde in PBS for 20 min and permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature. Following washing in PBS and blocking by incubation in 5% (w/v) bovine serum albumin (BSA) in PBS (buffer A), cells were incubated overnight with anti-LOX (ab174316, 1:100, Abcam, Cambridge, MA, USA) and anti-LOXL2 (ab96233, 1:100, Abcam) antibodies diluted in buffer A. Following washing, the cells were incubated for 1 h in Alexa Fluor 488 goat anti-rabbit secondary antibodies (A11070, 1:200 for LOX and 1:1000 for LOXL2, Invitrogen, Carlsbad, CA, USA) diluted in buffer A. The nuclei were stained with 4′,6-diamidino-2-phenylindole. Fluorescence images were obtained using confocal microscopy (TCS SP8; Leica,Wetzlar, Germany).

To stain F4/80 and LIFR, BMDMs from OSMR-KO and WT mice were cultured on coverslips, stimulated with 100 ng/mL LPS (#L4391, Sigma‒Aldrich, USA) or PBS for 24 h after serum starvation for 12 h, fixed with 4% paraformaldehyde, permeabilized in 0.5% Triton X-100, and incubated with anti-F4/80 (#ab6640, Abcam, USA) and anti-LIFR (#22779-1-AP, Proteintech, China) antibodies at 4 °C overnight. The next day, the cells were washed and incubated with Alexa Fluor^® 568 goat anti-rat or Alexa Fluor^® 488 goat anti-rabbit secondary antibodies (#A-11077, #A32731, Invitrogen, USA) for 60 min at 37 °C. Nuclei were stained with DAPI (#S36939, Invitrogen, USA). The slides were observed and photographed using an Olympus DX51 fluorescence microscope (Tokyo, Japan) and analysed using Image-Pro Plus 6.0 software (Media Cybernetics, Bethesda, USA).

Rat Schwann cells were cultured in Dulbecco's Eagle medium, 10% fetal calf serum (Atlanta Biological), neuregulin (0.5 μg ml⁻¹, Sigma), forskalin (0.2 μg ml⁻¹, Sigma) and penicillin-streptomycin. Cells at passage 22-29 were seeded onto 24-well dishes (Sigma) and incubated with collagen IV, nidogen-1 and different laminin constructs. Additionally, cells were treated with 28-fold excess compared with laminin of Net4-FL, Net4-FL^E195A,R199A at 37^oC for 1 h. Schwann cells were rinsed with PBS and fixed in 3% paraformaldehyde for 20 min followed by blocking with 5% goat serum in PBS overnight at 4 °C. Cells were incubated with anti-laminin γ1 (1:200, Millipore, MAB1920) and anti-collagen IV (10 μg ml⁻¹, Millipore, AB756P) at room temperature for 1 h. Alexa Fluor 647 goat anti-mouse and Alexa Fluor 488 goat anti-rabbit secondary antibodies (Molecular Probes) were used at 1:100 and counterstained with 4′,6-diamindino-2-phenylindol. Digital images of protein immunofluorescene levels were recorded with IPLAB 3.5 software (Scanalytics) and quantified with IMAGE J. Validation information for commercial antibodies is available on the manufacturers' websites. All experiments were performed in groups of five independent cultures and repeated at least twice.

Immunofluorescence microscopy was performed essentially, as described^{67 (link)}. Cells were grown to exponential phase and fixed with 1.6–2.6 % (w/v) paraformaldehyde and 0.008 % (w/v) glutaraldehyde. After permeabilization in GTE buffer (20 mM Tris/HCl, pH 7.6, 50 mM glucose, 10 mM EDTA), the fixed cells were incubated with suitable antibodies in PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄) containing 2 % (w/v) bovine serum albumin (BSA; Carl-Roth, Germany). First, target proteins were labeled with a polyclonal anti-BacO or anti-BacP^{49 (link)} antibody or a monoclonal anti-HA antibody (Millipore) at dilutions of 1:500, 1:400, and 1:200, respectively. Immunocomplexes were then visualized with Alexa-Fluor 594 Goat Anti-Rabbit or Alexa-Fluor 488 Goat Anti-Rabbit secondary antibodies (Molecular Probes) at a dilution of 1:200. Before imaging, SlowFade® Antifade (Invitrogen) was applied to each sample.

Co-cultured cells in suspension were fixed and permeabilized as above. Normal goat serum (10%) was applied for 1 h at room temperature to block cells, followed by incubation with primary antibodies at room temperature for 1 h. Primary antibodies included mouse monoclonal IgG anti-human β-catenin-APC (R&D Systems, Inc.) and rabbit polyclonal anti-GFP (Abcam, Inc., Cambridge, UK). Cells were washed twice and incubated with Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rabbit secondary antibodies (1:400; Molecular Probes, Life Technologies, Carlsbad, CA, USA) for 2 h. Negative controls consisted of secondary antibody application alone. Cells were washed twice, and nuclei were stained with 300 nm DAPI (Molecular Probes, Life Technologies) in PBS for 10 min, followed by a final wash and mounting in 100% glycerol. Confocal imaging was performed using a Leica laser-scanning confocal microscope (DMI 6000 SP8; Leica Microsystems, Wetzlar, Germany).

Tissue paraffin sections (5 μm) were mounted on slides and placed in a 55°C oven for 10 min, deparaffinized in xylene (3x, 5 min), hydrated using an alcohol series, 100%, 95%, and 70% alcohol (each 3x, 5 min), and rinsed in water. The sections were processed for antigen retrieval by boiling the slides in 10 mM Citrate Buffer (pH 6.0). The slides were cooled at room temperature for 20 min, washed in PBS, and blocked in 10% normal serum overnight at 4°C. Immunofluorescence was performed on serial sections from each group using rabbit anti-bax and anti-bcl-2 antibodies (Proteintech, USA). The sections were incubated with primary antibodies for 1 h at room temperature. Subsequently, sections were stained with Alexa Fluor® 488 goat anti-rabbit secondary antibodies (Molecular Probes, Inc.). The fluorescent-stained sections were watched by Laser Confocal Scanning Microscope (FV1000S-SIM/IX81, OLYMPUS, JAP) and analyzed by Software Image J to calculate as the average intensity of the fluorescent signal. Six visual fields from each slide were imaged and quantified to assess the final mean fluorescent signal for each sample.