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2 protocols using anti lamp

1

Chemically Induced Colitis Protocol

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LPS (Escherichia coli 0111:B4) was from Sigma-Aldrich. Dextran sulfate sodium (DSS, MW 47,000; MP Biomedicals) was purchased from MP Biomedicals. Antibodies against phospho-AKT473, phospho-ERK1/2, phosphor-STAT1, AKT, ERK1/2, STAT1, GAPDH were purchased from Cell Signaling Technology, PHLPP was from EMD Millipore, β-ACTIN was from Santa Cruz. FITC conjugated anti-mouse LY6G antibody, Alexa Fluor 647 conjugated anti-mouse CD182 (CXCR2) Antibody, APC/Cy7 and PE conjugated anti-mouse/human CD11B antibody were from Biolegend. ELISA Kit for TNFα, IL-1β, and IL-6 were from eBioscience, respectively. MPO ELISA Kit from R&D system. leukotriene B4 (LTB4), N-Formyl-Met-Leu-Phe (fMLF), and percoll were purchased from Sigma-Aldrich. EasySep™ Mouse Neutrophil Enrichment Kit was purchased from Stemcell Technologies. MIP-1α, fibronectin, and G-CSF was from Peprotech. Anti-PMP70 was from ThermoFisher Scientific. Anti-LAMP was from Abcam.
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2

Immunohistochemical Analysis of Retinal Cells

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Enucleated eyes were fixed in 4% paraformaldehyde at room temperature for 3 h, the anterior segment, and the posterior portion of the eye post-fixed in the same fixative overnight at 4°C before being placed in 25% sucrose for a second overnight period at 4°C for cryoprotection. Eyecups were embedded in OCT compound (Sakura Finetec, Torrance, CA) and cut into 10-μm thick sections. Primary antibodies (1:50) were: anti-LC3-II (MBL International, Japan), anti-LAMP (Abcam, Cambridge, MA), and biotin-conjugated mouse monoclonal anti-Thy-1 (BD-Pharmingen, San Jose, CA). Sections were exposed to the appropriate secondary antibodies: goat anti-mouse IgG FITC-conjugate (1:500; Southern Biotechnology, Birmingham, AL), or goat anti-rabbit IgG fluorescein-conjugate (1:500; Invitrogen, Carlsbad, CA). Anti-fade mounting media containing DAPI (EMC Biosciences, La Jolla, CA) was applied and sections cover-slipped.
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