Lungs of Wt and Pla2g5-null mice were excised and immersed in RPMI. Within 1h of surgery, the tissue was removed from RPMI and fixed in 4% paraformaldehyde, then embedded in Tissue-Tek® O.C.T.™ Compound (Sakura Finetek), and kept at −80°C until sectioning. Sections of 5-μm thickness were freshly cut, thaw-mounted onto slides, and stained for confocal microscopy. Frozen sections were rehydrated for 1h at RT then blocked with 10% donkey serum, followed by incubation with goat polyclonal IL-33 (AF3626, R&D Systems) and rabbit polyclonal proSPC (AB3786, Millipore, Temecula, CA) antibodies or appropriate isotypes controls at 4°C, overnight. Samples were washed, incubated at RT for 1h with appropriate secondary antibodies, washed and covered with Fluoroshield mounting media (Electron Microscopy Sciences, Hatfield, PA). Sections were imaged using a Nikon C1 plus laser scanner confocal system with a 40× oil Plan-Fluor NA1.3 objective lens. 8–10 Z-stack images of 0.5 μm were acquired through a small pinhole using Nikon EZ-C1 software. Images were analyzed using Image J (U.S. National Institute of Health, Bethesda, MD).
Fluoroshield mounting media
Manufactured by Electron Microscopy Sciences
Fluoroshield mounting media is a specialized liquid used to preserve and protect fluorescently labeled samples for microscopy applications. It is designed to minimize fading of fluorescent signals and maintain the structural integrity of the sample.
Automatically generated - may contain errors
2 protocols using fluoroshield mounting media
1
Characterizing IL-33 Expression in Lungs
2
Characterizing IL-33 Expression in Lungs
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Lungs of Wt and Pla2g5-null mice were excised and immersed in RPMI. Within 1h of surgery, the tissue was removed from RPMI and fixed in 4% paraformaldehyde, then embedded in Tissue-Tek® O.C.T.™ Compound (Sakura Finetek), and kept at −80°C until sectioning. Sections of 5-μm thickness were freshly cut, thaw-mounted onto slides, and stained for confocal microscopy. Frozen sections were rehydrated for 1h at RT then blocked with 10% donkey serum, followed by incubation with goat polyclonal IL-33 (AF3626, R&D Systems) and rabbit polyclonal proSPC (AB3786, Millipore, Temecula, CA) antibodies or appropriate isotypes controls at 4°C, overnight. Samples were washed, incubated at RT for 1h with appropriate secondary antibodies, washed and covered with Fluoroshield mounting media (Electron Microscopy Sciences, Hatfield, PA). Sections were imaged using a Nikon C1 plus laser scanner confocal system with a 40× oil Plan-Fluor NA1.3 objective lens. 8–10 Z-stack images of 0.5 μm were acquired through a small pinhole using Nikon EZ-C1 software. Images were analyzed using Image J (U.S. National Institute of Health, Bethesda, MD).
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