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3D-imaging reveals conserved cerebrospinal fluid drainage via meningeal lymphatic vasculature in mice and humans
Serial cross sections (5µm thick) were immunostained with rabbit anti-mouse LYVE1 (1:100) polyclonal antibody (11-034, AngioBio Co). .. DAB (3,3′ -Diaminobenzidine) staining was performed using the biotin avidin complex kit (PK-6100, Vectastain®Vector).
Efficacious, safe, and stable inhibition of corneal neovascularization by AAV-vectored anti-VEGF therapeutics
For analysis of corneal angiogenesis and lymphangiogenesis, the tissues were washed by 0.3% Triton X-100 in PBS and blocked with blocking buffer (0.3% Triton X-100/5% normal bovine serum albumin [BSA]; Cell Signaling Technology)/1× PBS for 1 h. The corneas were stained overnight at 4°C with rat anti-CD31 (PECAM-1, 1:400, sc-18916; Santa Cruz, Santa Cruz, CA, USA), rabbit anti-mouse LYVE-1 (1:200, 11-034; AngioBio), goat anti-mouse Dll4 (1:40, AF1389; R&D Systems), or rat anti-mouse CD11b (1:50, #550282; BD Pharmingen).
Sialoglycans on lymphatic endothelial cells augment interactions with Siglec-1 (CD169) of lymph node macrophages.
The tissue sections were then blocked with Carbo- Free Blocking solution (Vector Laboratories), followed by a second blocking step using the Streptavidin/Biotin Blocking Kit (Vector Laboratories) according to the manufacturer’s instructions. .. Primary antibodies and lectins were used as follows: polyclonal chicken anti- GFP (1:100, Aves Labs, Tigard OR, USA) for Ackr4- GFP, rabbit anti- mouse Lyve- 1 (1:300, AngioBio, San Diego, CA, USA), rat anti- mouse/ human PNAd (1:200, MECA- 79, BioLegend), biotinylated MAL- I (1:100, Maackia amurensis I, Vector Laboratories), biotinylated MAL- II (1:20, Maackia amurensis I, Vector Laboratories), biotinylated PNA (1:200, Arachis hypogaea, Sigma Aldrich).
In Vivo Lymphatic Imaging of a Human Inflammatory Breast Cancer Model
For LYVE-1 detection, following paraffin removal and antigen retrieval using citrate buffer pH 6, tissues were blocked with 5% bovine serum albumin (BSA) and stained with rabbit anti-mouse LYVE-1 antibody (AngioBio) and biotin-anti rabbit secondary antibody (Vector Labs). .. LYVE-1 expression was examined at x40 magnification (Leica Microsystems Inc.).

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