Tcs sp uv confocal microscope

CD4⁺, FOXP3⁺, and IL-17⁺ cells were detected in muscle biopsy samples by immunohistochemistry. Acetone-fixed, frozen sections were exposed to monoclonal antibodies against CD4 (Dako clone MT-310), FoxP3 (eBioscience, clone 236A/E7), or IL-17 (R&D systems Clone AF-317-NA) overnight at 4°C. Positive staining was revealed by peroxidase reaction (Dako real™ Detection System (K5001; Dako)). Double immunofluorescence was used to analyse FoxP3⁺ and CD4⁺ cell colocalisation. Detection of mouse monoclonal anti-FoxP3 and rabbit polyclonal anti-CD4 (Abcam®) antibodies was performed using Alexa Fluor (AF) 555 goat anti-mouse IgG (L+H) and AF 488 goat anti-rabbit IgG (L+H) secondary antibodies. For control purposes, a mouse IgG1 isotype control was included in the protocol. Stained muscle sections were analyzed with a Leica TCS-SP (UV) confocal microscope at the MSSM-Microscopy Shared Resource Facility (Pitie-Salpetriere Hospital, Paris).

Arabidopsis protoplasts were isolated and transfected as described [67 (link)]. Visualization for localization studies was with a Leica TCS SP UV confocal microscope, GFP being imaged with a 488 nm laser, RFP with a 561 nm laser, and protoplasts with DIC optics. For analysis of the ethylene response, the reporter constructs GCC-LUC [40 (link)] and ERF1-LUC [42 (link)] were used. The UBQ10-GUS construct was used as an internal control. Transfected protoplast samples in culture dishes were placed in air-tight containers with or without 10 μL L⁻¹ ethylene for 6 h at 22 °C under dim light (5 μE⋅m⁻²⋅s⁻¹). The results are shown as the means of relative LUC activities from duplicate samples with error bars.