Protocol detail

Immunohistochemical Analysis of Neuroinflammation in Parkinson's Disease

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Immunohistochemistry (IHC) was performed on 30 μm thick serial brain sections. Primary antibodies and working dilutions are detailed in Supplementary Table 5. TH, Nissl, p-α-syn^ser129 experimental procedures and quantitative analysis were performed as previously described^{39 (link),40 (link)}. Microglia and astrocytes from the SNpc region were stained with anti-Iba-1 (Wako, St. Louis, MO, USA) or anti-GFAP (Dako, Santa Clara, CA, USA) antibodies, followed by incubation with biotin-conjugated anti-rabbit antibody and ABC reagents. Sections were developed using SigmaFast DAB Peroxidase Substrate (Sigma-Aldrich). The number of microglia and densities of astrocytes in the SNpc region were measured with ImageJ software.

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Yun S.P., Kam T.I., Panicker N., Kim S., Oh Y., Park J.S., Kwon S.H., Park Y.J., Karuppagounder S.S., Park H., Kim S., Oh N., Kim N.A., Lee S., Brahmachari S., Mao X., Lee J.H., Kumar M., An D., Kang S.U., Lee Y., Lee K.C., Na D.H., Kim D., Lee S.H., Roschke V.V., Liddelow S.A., Mari Z., Barres B.A., Dawson V.L., Lee S., Dawson T.M, & Ko H.S. (2018). Block of A1 astrocyte conversion by microglia is neuroprotective in models of Parkinson’s disease. Nature medicine, 24(7), 931-938.

Publication 2018

anti-Iba-1 anti-GFAP SigmaFast DAB Peroxidase Substrate

Anti antibody Antibodies Astrocytes Biotin Brain Dilutions Gfap Immunohistochemistry Microglia Peroxidase Rabbit

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Variable analysis

independent variables

30 μm thick serial brain sections

dependent variables

Number of microglia
Densities of astrocytes in the SNpc region

control variables

Primary antibodies and working dilutions
TH, Nissl, p-α-syn^ser129 experimental procedures and quantitative analysis

Annotations

Based on most similar protocols

Ensure primary antibodies and working dilutions are detailed in Supplementary Table 5, as mentioned in the Input protocol and Protocol 1.

Perform antigen retrieval by boiling sections in 10 mM sodium citrate buffer, as described in Protocol 2 and Protocol 4.

Block endogenous peroxidase activity by incubating sections in 3% H2O2 for 30 minutes, as mentioned in Protocol 2 and Protocol 4.

Use biotin-conjugated secondary antibody and ABC reagents for signal amplification, as stated in the Input protocol and Protocol 1.

Develop the sections using SigmaFast DAB Peroxidase Substrate (Sigma-Aldrich), as described in the Input protocol and Protocol 1.

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