C-Fos Immunohistochemistry in Mouse Brain

Mice were sacrificed under Euthasol then transcardially perfused with PBS with heparin. Brains were removed and drop fixed in 4% PFA for 48 hours. After fixation, brains were washed with PBS, cryoprotected with 30% sucrose, then frozen in O.C.T. compound (Sakura Finetek) and sliced (40μM) with a cryostat (Leica). Free-floating sections were maintained in PBS + Azide (0.02%) until further processing. Immunohistochemistry for c-fos (1:1000 dilution; Millipore) on free-floating sections was performed as previously described^{39 (link)}.

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Filiano A.J., Xu Y., Tustison N.J., Marsh R.L., Baker W., Smirnov I., Overall C.C., Gadani S.P., Turner S.D., Weng Z., Peerzade S.N., Chen H., Lee K.S., Scott M.M., Beenhakker M.P., Litvak V, & Kipnis J. (2016). Unexpected role of interferon-γ in regulating neuronal connectivity and social behavior. Nature, 535(7612), 425-429.

Publication 2016

O.C.T. compound cryostat c-fos

Azide Brains Dilution Frozen Heparin Immunohistochemistry Mice Sucrose

Corresponding Organization : University of Virginia

Other organizations : University of Massachusetts Chan Medical School

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Variable analysis

independent variables

Sacrifice method (Euthasol)
Perfusion method (transcardial perfusion with PBS and heparin)
Fixation method (drop fixed in 4% PFA for 48 hours)
Cryoprotection method (30% sucrose)
Tissue slicing (40μM cryostat sections)

dependent variables

C-fos expression (immunohistochemistry)

control variables

Mouse species
Tissue processing steps (washing with PBS, freezing in O.C.T. compound)
Immunohistochemistry protocol (as previously described)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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