Lyve 1 antibody

Growth factor-reduced Matrigel matrix was purchased from Corning (Bedford, MA, USA). FxCycle™ PI/RNase Staining Solution, H2DCFDA, DAF-FM diacetate, and DAPI were bought from Life Technologies Corporation (Eugene, OR, USA). Anhydrous sodium sulfide (Na₂S), Pierce BCA protein assay kit, RIPA buffer, protease and phosphatase inhibitor mini tablets, RevertAid RT Reverse Transcription Kit, PowerUp SYBR™ Green Master Mix, TRIzol Reagent and CD68 antibody were procured from Thermo Scientific (Rockford, IL, USA). Na₂S solution was prepared in molecular biology grade water. Human oxidized low-density lipoprotein (oxLDL) was acquired from Kalen Biomedical, LLC (Montgomery, MD, USA). Cell proliferation reagent WST-1 was obtained from Roche Diagnostics GmbH (Mannheim, Germany). Phospho-eNOS (Ser1177), phospho-AKT (Ser473), phospho-ERK1/2 (Thr202/Tyr204), total AKT, total ERK1/2, total eNOS, Ki67, and CD45 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). GAPDH antibody was procured from Santa Cruz Biotechnology (Dallas, TX, USA). LYVE-1 antibody was purchased from Abcam (Cambridge, MA, USA). CD31 antibody was obtained from R&D Systems, Inc. (Minneapolis, MN, USA). Ready-to-use intercept blocking buffer to block the nitrocellulose membranes was purchased from Li-Cor Biosciences (Lincoln, NE, USA).

Rabbit polyclonal lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) antibody was purchased from Abcam. Santa Cruz Biotechnology supplied the following rabbit polyclonal antibodies: JAK2, VEGF-C, CCL4, and CCR5, as well as β-actin diluted 1:3,000 and STAT3-specific mouse monoclonal antibodies (mAbs) diluted 1:1,000. Recombinant human CCL4 and VEGF-C were purchased from PeproTech. Recombinant human CCL4 contains the substitutions of histidine for arginine at the 22nd position of the sequence and of glycine for serine at the 47th position. Staff from PeproTech tested the biological activity of Recombinant human CCL4 for its ability to chemoattract human blood monocytes. This testing confirmed the biological activity of CCL4 and showed that this product has equivalent biological activity as a natural chemokine. Inhibitors of JAK2 (product ID: CAS 457081037) and STAT3 (product ID: C1889) were purchased from Calbiochem (San Diego, CA, USA). A TRIzol kit was purchased from MDBio Inc., and TaqMan one-step PCR Master Mix was purchased from Applied Biosystems. Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Gibco-BRL Life Technologies. miR-195-3p mimic and control miRNA were purchased from Invitrogen. Thermo Fisher Scientific Inc. provided the Thermo Scientific Pierce BCA Protein Assay Kit. All other chemicals were purchased from Sigma-Aldrich.

CLs were fixed with 4% (v/v) paraformaldehyde (PFA) in PBS. 4 µm sections were mounted on glass slides pre-coated with silane (S3003; Dako, Glostrup, Denmark), deparaffined and rehydrated. Antigen retrieval was achieved by Tris-EDTA buffer pH 9.0 using microwave for 15 min at 600 W. Sections were immersed in methanol with 3% (v/v) H2O2 for 30 min and incubated with 10% (v/v) normal horse serum (MP-7500; Vector Laboratories Inc, Burlingame, CA, USA) for blocking. Then, sections were incubated with LYVE-1 antibody (ab33682; abcam, Cambridge, UK) as a specific marker of lymphatic endothelial cells [10] (link), [15] (link), [16] (link) diluted at 1∶4000 with PBS for 1 h at room temperature (RT). Negative control sections were incubated with normal rabbit serum diluted by PBS. Subsequently, sections were incubated with ImmPRESS UNIVERSAL reagent, anti-mouse/rabbit Ig (MP-7500; Vector Laboratories Inc, Burlingame, CA, USA) for 30 min according to the manufacturer’s instructions. The staining was visualized with 0.05% (w/v) 3,3′-diaminobenzidine (343-00901; Dojindo, Kumamoto, Japan) containing 0.01% (v/v) H2O2 and then counter-stained with hematoxylin. Bright field images were captured using FSX100 (Olympus, Tokyo, Japan) and merged using cellSens (Olympus, Tokyo, Japan).

Collected tissue samples from mice were fixed with 4% paraformaldehyde for 48 h, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) and Safranin O. Mice tissue sections were also processed as above with F4/80 (Abcam) and LYVE-1 antibody (Abcam) after deparaffinization using Clear Plus (Falma) without permeabilization. All samples were observed with a BZ-X810 (Keyence).

Formalin-fixed corneas from each group of animal were embedded in paraffin and 4 μm sections were prepared for examination. To access lymphangiogenesis, corneal sections were stained with and anti-mouse lymphatic vessel endothelial hyaluronan receptor (LYVE)-1 antibody (1:500; Abcam, Eugene, OR, USA) for 16 h at 4°C. After three washes with PBS for 15 min, the sections were then stained with a Texas Red-conjugated secondary antibody (Abcam). To detect F-actin, corneal sections were incubated with rhodamine-conjugated phalloidin (dilution 1:500, Abcam) for 1 h and washed three times with PBS. The stained sections were incubated with hoechst solution to stain nucleus before examined by fluorescence microscopy at 100× magnification.

After fixation in 10% formaldehyde, tissues were embedded in paraffin, and 3‐µm‐thick sections were used for immunostaining. The sections were incubated at 4°C overnight with a polyclonal rabbit anti‐CD31 antibody (Abcam) or a polyclonal rabbit anti‐lymphatic vessel endothelial hyaluronan receptor (LYVE)‐1 antibody (Abcam) as described previously.¹⁴