IHC was performed on striatal sections as described in our previous publications (6, 8, 100). Striatal sections (30 μm) were used for IHC. Citrate buffer was used to perform antigen retrieval [10 mM sodium citrate (pH 8.5)]. After antigen retrieval, sections were washed with PBS, blocked with blocking buffer (2% bovine serum albumin, 0.5% Triton X-100, and 0.05% Tween 20), and incubated in primary antibodies overnight at 4°C. Next, sections were washed with PBS, incubated in secondary antibodies for 1 hour, and stained with the nuclear dye Hoechst. Lastly, sections were mounted on precoated slides and dried overnight before visualizing them under a microscope. Confocal imaging was performed on these sections at the Iowa State University Microscopy Facility using a Leica DMEIR2 confocal microscope with 63× oil objective. For z-stacking, each section consisted of 10 to 15, 0.5-μm slices.

For ICC studies on microglial cells and microglial primary culture, 4% paraformaldehyde was used to fix the cells. Next, fixed cells were washed with PBS, blocked using blocking buffer, and incubated in primary antibodies following the manufacturer’s protocol. After primary antibody incubation, cells were washed with PBS, incubated in secondary antibody, and mounted on slides using Fluoromount aqueous mounting medium (Sigma-Aldrich). Samples were visualized using an inverted fluorescence microscope (Nikon TE2000-U). The primary antibodies used were the following: IBA1 (1:1000; AB_2314667, Wako), IBA1 (1:500; AB_870576, Abcam), Mul1 (1:500; AB_1860863, Abcam), NLRP3 (1:500; AB_2490202, AdipoGen Life Sciences), ASC (1:500; AdipoGen Life Sciences), Nos2 (1:500; AB_2152867, Santa Cruz Biotechnology), VPS35 (1:500; AB_2215220, Santa Cruz Biotechnology), and Mfn2 (1:500; Cell Signaling Technology). Alexa Fluor dye–conjugated secondary antibodies were used for ICC and IHC experiments.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.