Anti rat igg alexa fluor 568

Embryos or embryonic brains were dissected, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 10 μm. Immunofluorescent stainings were performed according to standard protocols. For antigen retrieval the sections were immersed in Tris-EDTA-Buffer pH 9 (10 mM Tris, 1 mM EDTA, 0,05% Tween-20) and treated in a Silit Sicomatic t-plus pressure cooker (2 rings, 10 min). The following primary antibodies were used: mouse anti-neurofilament 2H3 (Developmental Studies Hybridoma Bank, Iowa City, 1:100), chicken anti-GFP (Abcam Ab13970, 1:100), mouse monoclonal antibody “4F2” anti-LHX1 and LHX5 (Developmental Studies Hybridoma Bank, Iowa City, 1:100), mouse Ki67 (Becton Dickinson 550609, 1:100), mouse anti-ßIII-tubulin (Sigma-Aldrich T8660, 1:100), rat anti-BrdU (Abcam Ab6326, 1:100), mouse anti-BrdU/IddU (Becton-Dickinson 340649, 1:100), and rabbit anti-active Caspase 3 (Abcam Ab13847, 1:300). Secondary antibodies: anti-chicken IgG Alexa Fluor 488 (Life Technologies, 1:300), anti-rabbit IgG Alexa Fluor 488 (Life Technologies, 1:300), anti-mouse IgG Alexa Fluor 488 (Life Technologie, 1:300), anti-rat IgG Alexa Fluor 568 (Life Technologies, 1:300), and anti-mouse IgG Alexa Fluor 594 (Life Technologies, 1:300). Cell nuclei were stained with DAPI (Roth, 1:10000) or TO-PRO-3 (Life Technologies, 1:2000).

Lung and FALC were prepared by fixation in 4% paraformaldehyde at 4 °C for 1 h, incubating in 20% sucrose overnight for cryoprotection, and embedding in 15% sucrose/7.5% gelatin diluted in PBS. Overall, 12 μm frozen sections were cut using a Leica CM 3050 S cryostat. Sections were pre-blocked/permeabilized in PBS containing 3% BSA and 0.05% Triton X-100 and stained with primary antibodies anti-mCherry (M11217, Invitrogen), anti-Relmα (500-P214, PeproTech), or anti-actin α-smooth muscle FITC conjugated (1A4, Sigma), followed by secondary antibodies anti-rat IgG Alexa Fluor 568 (A11077, Life Technologies) and anti-rabbit IgG Alexa Fluor 488 (A21206, Life Technologies) for 1 h at the room temperature, or directly conjugated CD3 (clone 145-2C11), B220 (clone RA3-6B2), and CD169 (clone 3D6.112). Image acquisition was performed using either a Zeiss LSM 780 confocal microscope and ZEN 2010 acquisition software or a Nikon HCA fluorescence microscope and NIS-Elements 4.30.01 acquisition software for live cell imaging. Adobe Photoshop 11.0.2 was used to adjust brightness, contrast and colour balance (changes were applied to all images in equal measure).