Coverslips were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) for 20 min at room temperature and rinsed with PBS. Cells were blocked with 5% normal donkey serum (Millipore) and permeabilized in 0.2% Triton X-100 (Sigma) for 30 min at room temperature. Cells were then incubated in primary antibody solution for 1 h, rinsed with PBS and incubated in secondary antibody solution for 1 h at room temperature. Finally, nuclei were labeled with Hoechst nuclear stain (Sigma) to label DNA and mounted onto glass slides using FluoroMount medium (SouthernBiotech). Primary antibodies used were mouse anti–SMI-32 (Covance SMI-32R; 1:1000) and rabbit anti-mCherry (ThermoFisher; 1:1000). Secondary antibodies used were donkey anti-rabbit Cy3 (Jackson Immunoresearch 711–165–152) and donkey anti-mouse AF488 (Invitrogen A21202).
Fluoromount medium
Manufactured by Southern Biotech
FluoroMount medium is a liquid mounting medium designed for the preservation and visualization of fluorescently-labeled specimens. It is formulated to maintain the fluorescent signal and prevent fading.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (1 mentions)
Rabbit anti mcherry, by Thermo Fisher Scientific (1 mentions)
Donkey anti rabbit cy3, by Jackson ImmunoResearch (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Hoechst nuclear stain, by Merck Group (1 mentions)
S 11225, by Thermo Fisher Scientific (1 mentions)
L1516, by Merck Group (1 mentions)
Eclipse 90i microscope, by Nikon (1 mentions)
2 protocols using fluoromount medium
1
Immunofluorescence Staining of Neurons
2
Immunolabeling of Otx2 and WFA
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For immunostaining, 50 µm floating sections were incubated with anti-Otx2 (rat polyclonal, 1/200, in-house) and biotinylated-WFA (1/100, Sigma L1516) in TBS, 1% Triton-X, 0.2% Tween-20, 10% fetal calf serum, overnight at 4°C. Sections were extensively washed at RT, incubated 2 h at RT with anti-rat Alexa Fluor-488 (Molecular Probes A21208, 1/2,000) and streptavidin Alexa Fluor-546 (Molecular Probes S11225, 1/2,000), washed again and finally mounted in Fluoromount medium (SouthernBiotech). Images were acquired with an Eclipse 90i microscope (Nikon).
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