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Z033429

Manufactured by Agilent Technologies
Sourced in United States

The Z033429 is a laboratory instrument designed for conducting various analytical and testing procedures. It is a versatile piece of equipment that can be utilized in diverse research and testing applications. The core function of this product is to provide reliable and accurate measurements, data analysis, and sample processing capabilities to support scientific investigations and quality control processes. For detailed specifications and intended use, please refer to the product documentation or consult with an Agilent Technologies sales representative.

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4 protocols using z033429

1

Immunofluorescence Staining of Brain Sections

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Brains were processed through perfusion/fixation, as above. Serial 30 μm, free-floating coronal sections representing 1/6th of each brain were permeabilized in Tris-buffered saline containing Triton X-100 (TBST; 50 mM Tris with 0.9% NaCl, and 0.5% Triton X-100), incubated with 0.3% H2O2, and blocked with 10% horse serum/TBST. Primary antibodies were incubated in 1% horse serum/TBST for 48 h. Primary antibodies: glial fibrillary acidic protein (GFAP; 1:500, anti-rabbit, Dako Z033429), sex-determining region box 2 (Sox2; 1:100, anti-goat, Santa Cruz Biotechnology, sc-17320), Ki67 (1:500, Abcam ab15580), brain lipid binding protein (BLBP; 1:300, EMD Millipore, ABN14), doublecortin (DCX; 1:50, Santa Cruz Biotechnology, sc-8066, or 1:200 Abcam ab18723), FGF-23 (1:30, R&D Systems, MAB26291; 1:100, Mybiosource MBS2003657), cleaved caspase 3 (1:1000, Cell Signaling 9664), or S100β (1:400, Dako, Z0311). Primary antibody binding was visualized after incubation in fluorescently labeled secondary antibody (Life Technologies). Nuclei were labeled with 4’,6-diamidino-2-phenylindole (DAPI; Life Technologies) and mounted in Prolong Gold anti-fade mounting media (Life Technologies).
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2

Western Blot Analysis of Neuronal and Glial Markers

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Cells were lysed in RIPA buffer with protease inhibitor, and total protein was extracted and quantified (Pierce BCA Protein Assay Kit, Thermo Scientific). Total protein (20µg) was separated using a Bolt 4–12% Bis‐Tris Plus Gel (Life Technologies) and transferred to a nitrocellulose membrane using an iBlot2 dry blotting system (Thermo Scientific). Membranes were blocked for 1–4 h (Rockland Immunochemicals, VWR International, Arlington Heights, IL, USA); primary antibodies (rabbit anti‐Synapsin1, EMD‐Millipore AB1543P, 1:1,500; mouse anti‐PSD‐95, Neuromab, 1:1,500; chicken anti‐MAP2, Abcam ab5392, 1:2,000; rabbit anti‐GFAP, DAKO Z033429, 1:2,000) in blocking buffer incubated, shaking, overnight at 4°C and secondary antibodies (IRDye 680RD and IRDye 800CW, 1:5,000 in blocking buffer) for 1 h at room temperature. Proteins were detected by an Odyssey CLx infrared imaging system (LiCOR Biosciences, Lincoln, NE, USA), and semi‐quantitative analysis of signal intensity was corrected to the relative quantification of β‐actin.
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3

Immunohistochemical Staining of Neural Markers

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For immunohistochemical staining, rehydrated sections or coverslips were incubated with primary antibodies overnight. Primary antibodies used in this study were for Mettl3 (ab195352, 1:250, Abcam), neuronal nuclei (NeuN, MAB377, 1:500, Millipore), calbindin D-28K (C9848, 1:2,000, Sigma), and glial fibrillary acid protein (GFAP, Z033429, 1:1,000, Dako). Appropriate Horseradish peroxidase conjugated secondary antibodies (anti-Mouse IgG and anti-Rabbit IgG, Vector laboratories) were used according to manufacturer’s recommendations. A 3, 3′-diaminobenzidine kit (DAB, Vector laboratories) was used for color developing. Images were obtained using standard methods and imaged with a Leica Aperio VERSA 8 microscope (Leica Biosystems). Immunohistochemical images were analyzed with Image-Pro Plus (Media Cybernetics). At least three repeats were conducted for each calculation.
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4

Neurobehavioral Evaluation in BALB/c Mice

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Adult (2–3 months) BALB/c mice obtained from our in-house breeding colony were used in all experiments. ICV cannulation was performed under aseptic conditions as described previously58 (link). In brief, 4 μg/μL polybrene was incubated at 37 °C for 15 min, and ICV injection of 5 μL mixture was performed for ≥20 min.
At day 3 after ICV injection, Motor Performance Test-Accelerating Rotating Rod Test was performed. The rotating rod apparatus was used to evaluate motor function. The mice were placed on the rod (3 cm in diameter) for four trials. Each trial lasted for a maximum of 5 min, during which the rotating rod underwent linear acceleration from 0 to 30 rpm and then remained at maximum speed. Animals were scored for their latency to fall (in seconds) in each trial.
Then the mice were deeply anesthetized and euthanized by transcardiac perfusion with fixative solution (4% paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, 50 mL/animal). The brains were harvested and postfixed in 4% paraformaldehyde, dehydrated in increasing concentrations of sucrose (30–60%), infiltrated in OCT Tissue Freezing Medium (380148, Leica), and plastic-embedded. Frozen sections (15 μm) were cut, mounted on coverslips, and then dried at room temperature and stained with NeuN (ab128886, Abcam, 1:500) and GFAP (Z033429, Dako, 1:1000).
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