For the myenteric plexus, fresh gastric tissues were placed in pre-cooled phosphate-buffered saline (PBS) solution. Thereafter, the mucosa and muscularis tissues were separated and fixed in 4% paraformaldehyde for 10 min. The paraffin-embedded gastric tissue sections were dewaxed and hydrated and subjected to antigen retrieval. The tissues were incubated with donkey serum containing 0.3% Triton X‐100 at 4 °C overnight for blocking of nonspecific binding. Subsequently, the tissue sections were incubated overnight at 4 °C with the specific primary antibodies: GFAP (ABclonal, Wuhan, China), HuC/D (Abcam, Cambridge, UK), S100B (Abcam, Cambridge, UK), β-Tubulin (ABclonal, Wuhan, China) H3K9me1 (A2358, ABclonal), H3K9me3 (A2360, ABclonal), H3K27me1 (A2361, ABclonal), and H3K27me3 (A2363, ABclonal). After washing three times with PBS, the preparations were then stained with the secondary antibody and incubated for 2 h at room temperature. The cell nuclei were stained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) for 20 min. The specimens were observed using a confocal laser scanning microscope (Olympus, Tokyo, Japan).
Glial fibrillary acidic protein (gfap)
Manufactured by ABclonal
Sourced in United States
GFAP is a protein that is specifically expressed in astrocytes, a type of glial cell in the central nervous system. It is commonly used as a biomarker for astrocyte activation and injury.
Automatically generated - may contain errors
Lab products found in correlation
H3k27me3, by ABclonal (2 mentions)
H3k9me1, by ABclonal (2 mentions)
H3k27me1, by ABclonal (2 mentions)
H3k9me3, by ABclonal (2 mentions)
Iba 1, by Abcam (2 mentions)
Confocal laser scanning microscope, by Olympus (1 mentions)
Huc d, by Abcam (1 mentions)
β tubulin, by ABclonal (1 mentions)
Fbxo3, by Santa Cruz Biotechnology (1 mentions)
Hipk2, by Santa Cruz Biotechnology (1 mentions)
Coralite594 conjugated goat anti rabbit igg antibody, by Proteintech (1 mentions)
Antifade mounting medium with dapi, by Beyotime (1 mentions)
Il 10, by ABclonal (1 mentions)
Interleukin 6 (il 6), by ABclonal (1 mentions)
Gapdh, by Antgene (1 mentions)
Ecl kit, by Vazyme (1 mentions)
Bca protein assay kit, by Vazyme (1 mentions)
Aperio slide scanner, by Leica (1 mentions)
5 protocols using glial fibrillary acidic protein (gfap)
1
Myenteric Plexus Immunofluorescence Imaging
2
Immunofluorescence Analysis of Brain Sections
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Brain sections (3 μm thickness) were dewaxed and placed in a box with an EDTA antigen repair buffer (PH 8.0), then put in a microwave to repair the antigen. Meanwhile, the coverslips of HT22 cells were fixed, permeabilized, and blocked. The primary antibodies, including NeuN (1:50, ABclonal, A19086), Iba-1 (1:50, Abcam, Boston, MA, USA, ab178847), GFAP (1:50, ABclonal, A19058), FBXO3 (1:50, Santa Cruz Biotechnology, sc-514625; 1:50, CUSABIO, CSB-PA892132LA01HU), and HIPK2 (1:50, Santa Cruz Biotechnology, sc-100383), along with MPO (1:50, Servicebio, Wuhan, Hubei, China, GB11224) were used at 4 °C for 12 h. Subsequently, brain sections and coverslips were incubated with CoraLite488-conjugated Goat Anti-Mouse IgG antibody (1:200, Proteintech, SA00013-1) and CoraLite594-conjugated Goat Anti-Rabbit IgG antibody (1:200, Proteintech, SA00013-4) for 1 h away from light. Ten minutes after dropping Antifade Mounting Medium with DAPI (Beyotime Biotechnology, Shanghai, China) on the coverslips and sections, visualization was accomplished with a laser scanning confocal microscope (LSCM).
3
Perioperative Cerebral Oxygenation and Biomarkers
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Blood samples from the radial artery were collected immediately before induction of anesthesia (T0), 10 min (T1) and 60 min (T2) after turning over, immediately after the operation (T3), and 15 min after extubation (T4). Blood gasses were analyzed; pH, PaO2, PaCO2, and SaO2 were recorded; and PaO2/SaO2 was calculated. Simultaneously, rSO2 and the incidence of cerebral desaturation during surgery were recorded. Serum concentrations of interleukin (IL)-6, IL-10, and glial fibrillary acidic protein (GFAP) were determined by commercial IL-6 (ABclonal, Woburn, MA, USA), IL-10 (ABclonal) and GFAP (CUSABIO, Houston, TX, USA) ELISA kits, respectively.
4
Protein and Histone Extraction and Analysis
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The total proteins and nuclear proteins were extracted from the cells or tissue samples using a radioimmunoprecipitation assay (RIPA) buffer supplemented with phenylmethylsulfonyl fluoride (PMSF). Protein concentration was determined using bicinchoninic acid (BCA) protein assay kit (Vazyme, China). The proteins were then separated using SDS-PAGE and then electro-transferred to polyvinylidene difluoride (PVDF) membranes. After soaking in 10% skimmed milk powder for 1 h, the membranes were incubated overnight at 4 °C with specific antibodies: GFAP (ABclonal, Wuhan, China), GDNF (Abcam, Cambridge, UK), S100B (Abcam, Cambridge, UK), GAPDH (Antgene, Wuhan, China), H3K9me1 (A2358, ABclonal), H3K9me3 (A2360, ABclonal), H3K27me1 (A2361, ABclonal), H3K27me3 (A2363, ABclonal), and H3 (A2348, ABclonal). Thereafter, the membranes were incubated at room temperature with HRP-labeled secondary antibodies for 1 h. The intensities of the protein bands were determined using a chemiluminescence (ECL) kit (Vazyme).
5
Multimodal Histopathological Analyses
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The tibialis anterior muscle, distal colon, lumbar spinal cord, and brain were post-fixed in 10% neutral buffered formalin for 48 h and then switched to 70% ethanol until ready for embedding with paraffin. Paraffin embedded samples were cut into 6-8um sections on slides for staining. Tissue sections were deparaffinized, rehydrated and probed with the following antibodies: hSOD1 (SOD1 MS785, GTX57211), ChAT (ProteinTech, 20,747-1-AP), GFAP (ABclonal, A10873), Iba1 (Abcam, ab17886), Histochemistry was performed using antigen retrieval with citric acid buffer at 95C for 30 min and Vector ABC and NovaRed substrate system according to manufacturer instructions (Vector Laboratories). Please refer to the table for additional antibody information. Luxol Fast Blue (LFB) and eosin stain of the spinal cord were performed by the Dept. of Pathololgy at University of Louisville. Slides were scanned using an Aperio Slide Scanner (Leica Biosystems) with a 20X objective. Images were quantified in ImageJ using optical density, mean gray value, or % area depending on context, described in figure legends.
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