Mice (n = 6 in each group) were decapitated, the hippocampus were immediately dissected, frozen and stored at −80 °C. Tissue samples were lysed and western blots were performed as previously described [11 (link)]. Primary antibodies include GFP (1:1000, Beyotime, cat # AG281, Shanghai, China), Glial fibrillary acidic protein (GFAP) (1:1000, Wanleibio, cat # WL0836, Shenyang, China), Flag (1:1000, abm, cat # G188, Zhenjiang, China), Oligo2 (1:1000, Wanleibio cat # WL04942, Shenyang, China), NeuN (1:1000, Wanleibio, cat # WL03099, Shenyang, China), doublecortin (Dcx) (1:1000, Proteintech, cat # 13925-1-AP, Rosemont, USA), GAPDH (1:5000, Boster, cat # BM1623, Wuhan, China) and β-Tubulin (1:5000, Beidibio, Nanjing, China). HRP-conjugated secondary antibodies purchased from Immunoway Biotechnology Company (goat anti mouse cat # RS0001 and goat anti rabbit cat # RS0002, Plano, TX, USA) were used to probe these blots. Protein visualization was carried out using an enhanced chemiluminescen (ECL, Meilunbio, Dalian, China). The signal intensity was obtained by densitometric scanning.
Glial fibrillary acidic protein (gfap)
Manufactured by Wanlei
Sourced in China
GFAP is a protein found in the cytoplasm of astrocytes, a type of glial cell in the central nervous system. It is widely used as a biomarker in various research and diagnostic applications.
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Lab products found in correlation
Gapdh, by Boster Bio (1 mentions)
Flag tag (flag), by Applied Biological Materials (1 mentions)
Hrp conjugated secondary antibodies, by Immunoway (1 mentions)
Enhanced chemi luminescence (ecl), by Meilun (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Cy3 goat anti rabbit igg, by ABclonal (1 mentions)
Fitc goat anti rabbit igg, by ABclonal (1 mentions)
Fluorescein isothiocyanate (fitc), by Boster Bio (1 mentions)
4 6 diamidino 2 phenylindole dapi, by Boster Bio (1 mentions)
4 protocols using glial fibrillary acidic protein (gfap)
1
Hippocampal Protein Analysis in Mice
2
Evaluating Astrocyte Hypertrophy in Spinal Cord
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Sections of the L4–6 spinal cord (paraformaldehyde fixed and paraffin embedded) were processed for immunohistochemistry using a primary antibody against glial fibrillary acidic protein (GFAP) (1 : 600, Wanleibio, China). Following incubation overnight at 4°C, sections were incubated in a horseradish peroxidase-conjugated secondary antibody for 4 h at room temperature. The color was developed with 3,3′-diaminobenzidine (DAB). The integral optical density (IOD) and area of hypertrophy in GFAP-positive cells were evaluated using IPP software.
3
Immunofluorescence Analysis of Brain Tissue
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Immunofluorescence staining was performed on sections of whole brain from three rats in each group. The brain tissues were preserved in a 4% paraformaldehyde solution at 4°C, then dehydrated and embedded in paraffin. Next, 4 μm-thick coronal sections were obtained. The sections were then deparaffinized, subjected to antigen retrieval, permeabilized with Triton X-100, washed with PBS, and blocked with 10% goat serum (Wolcavi Biotechnology Co., Ltd, Beijing, China). The sections were incubated overnight at 4°C with polyclonal rabbit antibodies to glial fibrillary acidic protein (GFAP; 1:200, Wanleibio, Shenyang, China, Cat# WL0836, RRID: AB2893014) or myelin basic protein (MBP; 1:200, ABclonal, Wuhan, Hubei Province, China, Cat# A1664, RRID: AB2763719). The sections were rinsed with PBS again and then incubated for 90 minutes at 37°C with Cy3 goat anti-rabbit IgG (1:500, ABclonal, Cat# AS007, RRID: AB2769089) or FITC Goat Anti-Rabbit IgG (1:500, ABclonal, Cat# AS011, RRID: AB2769476). The sections were then washed with PBS, stained with 4, 6-diamidino-2′-phenylindole (Sigma, St. Louis, MO, USA) for 15 minutes, sealed, washed with PBS, and viewed under a fluorescence microscope (Leica, Wetzlar, Germany).
4
Immunohistochemical Analysis of Klotho and Glial Activation in Mice
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Immunohistochemistry and fluorescence staining were performed to examine Klotho protein expression in the choroid plexus and glial activation in the hippocampal CA1 subregion in mice as previously described (Kuang et al., 2014a (link); Mao et al., 2017 (link)). The selected coronal sections were incubated with a respective antibody against Klotho (1:200; Transgene, Raleigh, NC, United States), glial fibrillary acidic protein (GFAP; 1:100; Wanleibio, Shenyang, Liaoning, China), or Iba-1 (1:500; Wako, Wakayama, Japan) at 37°C for 2 h and then at 4°C overnight. The sections were then incubated with a secondary antibody conjugated with or without fluorescein isothiocyanate (Boster Biological Technology, Wuhan, Hube, China), followed by nucleus counterstaining with hematoxylin or 4,6-diamidino-2-phenylindole (DAPI; Boster). For semiquantitative analysis of the immunostaining results, the choroid plexus or hippocampal CA1 region was digitized using a 40× objective. Immunoreactivity was determined based on the integrated optical density (IOD) of Klotho-positive immunostaining in the choroid plexus or the percent area of GFAP- and Iba-1-positive immunostaining in the CA1 subfield using Image Pro Plus 6.0.
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