GFAP expression in the mouse hippocampus was measured using immunohistochemistry (IHC). The mouse hippocampus was perfused through the heart with 4% paraformaldehyde. Paraffin sections were prepared and deparaffinized through dimethyl benzene and ethanol, and antigens were retrieved by incubation in a citric acid buffer (pH 6.0) at 100°C for 20 min. The sections were incubated with 3% H2O2 for 10 min to block any endogenous peroxidase activity and then with 5% BSA for 35 min to block non-specific binding. Primary anti-GFAP antibodies (ab68428, 1:400, Abcam) were then added and incubated at 4°C for overnight. After washing three times with PBS, the sections were incubated with the goat anti-rabbit IgG antibody (ab6721, 1:8,000, Abcam) for 35 min at 37°C. The sections were stained with the DAB Horseradish Peroxidase Color Development Kit and then dyed with hematoxylin. Finally, the sections were imaged using a microscope.
Ab68428
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab68428 is a primary antibody that targets the Vimentin protein. It is a polyclonal antibody produced in rabbit. The core function of this product is to enable the detection and analysis of Vimentin expression in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab78078, by Abcam (2 mentions)
Ab6721, by Abcam (1 mentions)
Anti pakt s473, by Cell Signaling Technology (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti cyclin d1, by Cell Signaling Technology (1 mentions)
Anti p s6 s235 236, by Cell Signaling Technology (1 mentions)
Ab44976, by Abcam (1 mentions)
Ab32199, by Abcam (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Anti nestin, by Abcam (1 mentions)
Anti p pten, by Cell Signaling Technology (1 mentions)
Anti s6, by Cell Signaling Technology (1 mentions)
Anti parp, by Cell Signaling Technology (1 mentions)
Eos digital camera, by Canon (1 mentions)
Ab7481, by Abcam (1 mentions)
Ab218011, by Abcam (1 mentions)
Ab228550, by Abcam (1 mentions)
Ffp24, by Beyotime (1 mentions)
Ab18207, by Abcam (1 mentions)
Ab194113, by Abcam (1 mentions)
19 protocols using ab68428
1
Measuring GFAP Expression in Mouse Hippocampus
2
Comprehensive Analysis of Neural Stem Cell Markers
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Anti-PTEN (ab32199, Abcam, USA), anti-p-PTEN (#9554, Cell Signaling Technology, USA), anti-glial fibrillary acidic protein (ab68428, Abcam), anti-βIII-tubulin (ab78078, Abcam), anti-Nestin (ab6142, Abcam), anti-cyclin-D1 (#2987, Cell Signaling Technology), anti-caspase-3 (ab44976, Abcam), anti-PARP (#9532, Cell Signaling Technology), anti-pS6(s235/236) (#4858, Cell Signaling Technology), anti-S6(#2317, Cell Signaling Technology), anti-pAKT(s473) (#4060, Cell Signaling Technology), anti-AKT (#2920, Cell Signaling Technology), anti-CGRP (#14959, Cell Signaling Technology), anti-β-actin (#3700, Cell Signaling Technology).
3
Quantitative Analysis of Spinal Cord Plaques
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Following deparaffinization and hydration, sections were selected randomly from the lumbar spinal cord and hippocampus. All sections were retrieved by pressure cooker and permeabilized by 20% tween for 20 min for fluorescence immunostaining. The nonspecific area was blocked with 0.1% BSA in 0.1% Triton X-100/PBS for 20 min. The sections were incubated overnight at 4◦ C with Anti-MBP antibody (1:500, Abcam [ab218011], USA) and Anti-GFAP antibody (1:800, Abcam [ab68428], USA) and Anti-IDO-1 Antibody (1:250, EMD Millipore [MABF850], Germany), as primary antibody, in 0.03% PBS-Triton- × 100 supplemented with 5% normal goat serum (Abcam[ab7481-], USA). The slides were incubated with appropriate secondary antibodies (Alexa Fluor 488 and 566 (1:500, Thermo Fisher Scientific, USA), and Hoechst (1:1000, Abcam [ab228550], USA) for 1 h. Samples were analysed using a fluorescent microscope (Olympus IX-71; Olympus, Tokyo, Japan) equipped with a Canon EOS digital camera. For quantification of plaque number, we used soteriological dissector method for particle number. after preparation of immunohistochemically imaging of spinal cord, two adjust section with mentioned distance compared them (position and plaque size). The plaque with different position were counted. Totally number of each spinal cord plaque calculated for comparison between groups. Area of plaque estimated image J software.
4
Protein Expression Analysis of Neural Stem Cells
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The C17.2 NSCs treated with or without RA were lysed on ice using RIPA buffer containing protease inhibitors (P8465, Sigma). The lysates were then quantified using a BCA protein assay kit (Pierce). Equal amounts of total protein (10 μg) were separated by electrophoresis on 12% Bis‐Tris gels and transferred onto a PVDF (polyvinylidene fluoride) membrane (FFP24, Beyotime, China). The membrane was subsequently probed with primary antibodies overnight at 4°C. The primary antibodies used were as follows: anti‐AKT (1:2000, 10,176‐2‐AP, Proteintech, USA), anti‐p‐AKT (1:2000, 66,444‐1‐Ig, Proteintech), anti‐β‐actin (1:5000, 66,009‐1‐Ig, Proteintech), anti‐GFAP (1:10000, ab68428, Abcam), anti‐β‐III tubulin (Tuj‐1) (mouse, 1:2000, ab18207, Abcam) and nestin (mouse, 1:500, Proteintech). The secondary antibodies used Goat anti‐Mouse IgG (H + L) Secondary Antibody (1:5000, AWS0001, Abiowell) And Goat anti‐Rabbit IgG (H + L) Secondary Antibody (1:5000, AWS0002, Abiowell). After washing, the membrane was incubated with a peroxidase‐conjugated secondary antibody for 2 h at room temperature. Immunoreactive bands were detected using an ECL kit following the manufacturer's instructions. Subsequently, the membrane was reprobed with anti‐β‐actin as an internal loading control.
5
Immunohistochemical Analysis of Neuroinflammation
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Frozen brain sections (12 μm thick) were incubated with rabbit anti-NeuN (ab177487, Abcam, Cambridge, MA, USA), goat anti-IL-33 (AF3626, R&D Systems, Minneapolis, MN, USA), rabbit anti-SULT2A1/ST2 (ab194113, Abcam, Cambridge, MA, USA), goat anti-GFAP (ab68428, Abcam, Cambridge, MA, USA), and rabbit anti-Iba1 (019-019741, Wako, Japan). Stained sections were incubated with fluorescent secondary antibody and mounted for nuclear labeling using fluorescence mounting medium containing DAPI. We used Roche In Situ Cell Death Detection Kit (11684817910) to perform TUNEL staining (fluorescein). After nuclear staining, the slices were visualized and imaged with a fluorescence microscope. Images were captured at ×20 magnification. The numbers of IL-33+, ST2+, GFAP+ and Iba1+ cells were counted and normalized to the area. Forty-five images (3 field/section, 3 sections/animal, n = 5) of each group were analyzed using ZEN software.
6
Immunohistochemical Analysis of Retinal Tissues
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Retinal tissues were cryoprotected in 30% sucrose for 24 h and embedded in OCT medium (Thermo Scientific, Cat# 6502). Ten-micrometer tissue sections were cut at -20°C in a cryostat (Thermo Scientific) and collected on the poly-L-lysine coated slides. After blocking with 5% BSA and 0.1% Triton X-100 in PBS, the retinal sections were incubated overnight at 4°C with the following primary antibodies: GFAP (1:200, Abcam Cat# ab68428, RRID: AB_1209224), NeuN (1:300, Abcam Cat# ab177487, RRID: AB_2532109), Calretinin (1:500, Santa Cruz Biotechnology Cat# sc-365956, RRID: AB_10846469), Calbindin (1:200, Santa Cruz Biotechnology Cat# sc-365360, RRID: AB_10841576), Rhodopsin (1:400, Abcam Cat# ab5417, RRID: AB_304874) and PKCα (1:400, Abcam Cat# ab32376, RRID: AB_777294). The retinal sections were washed and incubated for 3 h at room temperature with the fluorophore-conjugated secondary antibodies. The retinal sections were observed using an Olympus IX-73 microscopy and the fluorescent signals were analyzed by Image J.
7
GFAP Protein Immunoprecipitation and Western Blot
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Three hundred micrograms of cecum protein extract was incubated for 2 h at RT and then overnight at 4°C with rabbit monoclonal anti-GFAP antibody (1:40, Ab68428, Abcam, France). The immune complexes were captured with 50% Protein A Agarose beads (Cell Signaling, Saint Quentin Yvelines, France) on a rotator for 2 h at RT. After centrifugation (4°C, 20,000g, 5 min), the precipitates were washed three times with RIPA buffer. The final pellets were suspended in 30 µl of 2× loading buffer and incubated at 95°C for 15 min. The entire suspension was loaded onto a SDS-PAGE gel, and the Western blot for GFAP (1:10,000) was performed as described above and using peroxidase-labeled secondary anti-rabbit antibody (1:400, Abcam, France). Blot image analysis was performed as described above.
8
Immunohistochemical Analysis of Neural Markers
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The tissues were treated with 0.01 M citric acid for antigen retrieval. After washing with PBS, the slides were blocked with TBST containing 10 goat serum for 1 h. Then, the tissues were incubated with primary antibodies overnight at 4° C. The primary antibodies and dilution times were listed as follows. GAP43 (1:1000, ab232772, Abcam, UK), NF421 (1:800, ab187374, Abcam, UK), GFAP (1:1000, ab68428, Abcam, UK), Bax (1:800, ab81083, Abcam, UK), Cleaved Caspase-3 (1:1000, #961S, Cell signaling, USA), Bcl-2 (1:800, ab32124, Abcam, UK). Then, the sections were washed using PBS and incubated with related secondary antibodies for 2 h at room temperature. The secondary antibodies and dilution times were listed as follows. Goat anti-rabbit lgG (1:2000, ab205718, Abcam, UK) and Goat anti-mouse lgG (1:2000, ab205719, Abcam, UK). All sections were mounted and observed under a microscope (Leica, Germany). Image J software was used to quantify and normalize images.
9
Immunocytochemical Staining of Glial and Immune Cells
10
Immunofluorescence Analysis of Hippocampal Slices
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The brain tissues were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 24h at 4°C and then washed twice in PBS. The samples were then dehydrated through an ethanol series, cleared by soaking in xylene, embedded in paraffin, and sectioned into 4μm. Slides containing paraffin sections were deparaffinized in xylene and rehydrated through an ethanol series, then incubated in Citrate Antigen Retrieval Solution (Beyotime, P0081) at 90 °C for 20min. The sections were washed with PBS and incubated with 10% BSA for 2h and then were incubated with primary antibodies against synapsin I (AB1543P">AB1543P , Millipore), PSD95 (ab2723, Abcam), CXCL5 (PA5-103851, Invitrogen), GFAP (Ab68428, Abcam), and MAP2 (ab254264,Abcam) at 1:200 dilution in PBS containing 2% BSA overnight at 4°C. Subsequently, the specimens were incubated with anti-rabbit IgG conjugated to Alexa Fluor 488 (A-21206, Thermo Fisher) and anti-mouse IgG Alexa Fluor 594 (R37115, Thermo Fisher) at 1:400 dilution. Staining images were visualized using Leica confocal microscope (Lecia Microsystems Inc., Buffalo, USA). Images of hippocampal slice were transferred to ImageJ software and quantified by measuring the average pixel intensity of the hippocampus.
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