Anti-GFAP antibody ValidAbTM-100mug

Artikelnummer: HLB-HB8267
Artikelname: Anti-GFAP antibody ValidAbTM-100mug
Artikelnummer: HLB-HB8267
Hersteller Artikelnummer: HB8267
Alternativnummer: HLB-HB8267-100UG,HLB-HB8267-25UG
Hersteller: HelloBio
Wirt: Mouse
Kategorie: Antikörper
Applikation: ICC, WB
Spezies Reaktivität: Mouse, Rat
Immunogen: GFAP purified from porcine spinal cord
Alternative Synonym: glial fibrillary acidic protein, ALXDRD
Antibody to GFAP - cytoskeletal protein used as an astrocyte marker. Part of the ValidAb(TM) range of highly validated, data-ri
Klonalität: Monoclonal
Konzentration: 1mg/ml
Klon-Bezeichnung: [GA-5]
Isotyp: IgG1
NCBI: 2670
UniProt: P14136
Puffer: Lyophilised. When reconstituted contains PBS with 15mM sodium azide and 1% recombinant BSA
Expression System: GFAP is primarily expressed within astrocytes of the central nervous system alongside also expressing in non-myelinating Schwann cells of the peripheral nervous system and satellite cells of the peripheral ganglia. GFAP expression has also been reported
Reinheit: {no attribute purity}
Target-Kategorie: GFAP
Antibody Type: Primary Antibody
Application Verdünnung: WB: 1µg/ml (1:1000) as measured in rat brain cytosol. IHC(IF): 1µg/ml (1:1000) as measured in free-floating fixed hippocampal sections. ICC: 1µg/ml (1:1000) as measured in neuronal cell culture.
Figure 2. GFAP and Vimentin expression in a rat cultured neuron preparation visualised using HB8267 and an anti-Vimentin antibody. GFAP and Vimentin are both expressed in glial cells and can be seen to express at different levels in individual cells creating a colour gradient across the cell population. Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB8267 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) with a goat anti-Vimentin antibody being incubated at a 1:500 dilution (2µg/ml). Secondary antibodies (Polyclonal donkey anti-mouse DyLight 488 conjugated, Thermofisher SA5-10166, 1:300 dilution and Polyclonal donkey anti-goat DyLight 594 conjugated, Thermofisher SA5-10088, 1:300 dilution) were incubated for 1 hour at room temperature. For more detail please see our ICC protocol. Images were captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope. The image was captured in a tile scan and z-stack (0.49µm spacing) using Lightning adaptive deconvolution and a 63x objective. Lasers used were 405nm (20.0% power, PMT: 712.7V gain), 496nm (1.0% power, HyD: 14.1% gain) and 561nm (1.5% power, HyD: 10% gain). The tile scan was merged using LASX before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 1. GFAP expression in a rat cultured neurone preparation visualised using HB8267. GFAP expression in the dense intermediate filament network of glial cells was successfully visualised using HB8267 at a 1:1000 dilution (1µg/ml). Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB8267 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) followed by a one hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica SP5-II confocal laser scanning microscope coupled to a Leica DMI 6000 inverted epifluorescence microscope. The image was captured using a 63x objective (2x zoom), 405nm (20.0% power) and 488nm (20.0% power) laser lines in a z-stack (0.335 µm spacing). Deconvolution was carried out using Huygens Essential (Scientific Volume Imagine) followed by the stack being flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 3. GFAP labelled astrocyte in rat brain. HB8267 staining reveals an astrocyte in rat brain tissue surrounded by axonal processes labelled by HB7266 (rabbit monoclonal anti-neurofilament L). Method: Rat brains were dissected and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm transverse slices before sections were incubated in 1% NaBH4 for 30 minutes followed by 0.05M glycine for 30 minutes. Sections were blocked in 2% BSA, 3% goat serum before incubation overnight in HB8267 (1:1000 dilution, 1µg/ml) and HB7266 (1:2000 dilution, 0.5µg/ml). This was followed by a two hour incubation with secondary antibodies (polyclonal goat anti-rabbit DyLight 594 conjugated, Thermofisher 35561, 1:300 dilution and polyclonal goat anti-mouse Janelia Fluor 525 (HB8455) conjugated (1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol . The image was captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope. The image was captured using Lightning adaptive deconvolution using a 40x objective and 405nm (21.1% power, PMT: 592V gain), 561nm (0.5% power, HyD: 22.7% gain) and 514nm (1.0% power, HyD: 10.0% gain) lasers. Images were captured as a stack (0.49µm z-spacing) being flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 4. GFAP expression in various tissue lysates and preparations. HB8267 revealed a band of size 49kDa only present in brain derived samples. Method: mouse brain and rat brain membrane (P2) and cytosol fractions were prepared following previous work (Molnar et al., 1993. Neuroscience 53:307-326) from freshly collected adult brains. Other tissue lysates were prepared following established protocols from freshly dissected tissue (see our guide on WB sample preparation). Samples were loaded (20µg / lane) onto a 10% acrylamide gel alongside a protein ladder (Thermofisher, 26617) before being run at 60V for 40 minutes followed by 120V for 90 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. The membrane was blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB8267 at a 1:1000 dilution (1µg/ml). Following washing the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 10 min exposure, 700nm channel: 2 min exposure).
Figure 5. The effect of varying HB8267 concentration upon staining in rat hippocampus. HB8267 produced a strong signal to noise ratio at dilutions as low as 1in4000 (250 ng/ml). Method: hippocampi were dissected from rat brains and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) for another 24hrs. A freezing microtome was used to cut 40µm transverse slices before sections were incubated in 0.05M glycine for 30 minutes. Sections were blocked in 1% BSA, 22.52mg/ml glycine before incubation overnight in varying dilutions of HB8267 ranging from 1:500 (2µg/ml) to 1:4000 (0.25µg/ml) with a no primary step omitting the addition of HB8267 to the incubation buffer. This was followed by a two hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. Images were captured using a Leica DM2500 epifluorescence microscope (20x objective) coupled to a Leica DFC7000T colour digital camera with DAPI and I3 filters. Exposure times were as follows: 1:500 – DAPI 10x gain, 11ms exposure, I3 10x gain, 26.9ms exposure 1:1000 – DAPI 10x gain, 18.3ms exposure, I3 10x gain, 18.3ms exposure 1:2000 – DAPI 10x gain, 11.1ms exposure, I3 10x gain, 27.1ms exposure 1:4000 – DAPI 10x gain, 11.1ms exposure, I3 10x gain, 32.5ms exposure No primary – DAPI: 10.18ms exposure, I3: 112.ms exposure (taken using different microscope: Leica DMI6000B with Photometric-Prime95B camera). Images were processed in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682) using the subtract background (50px rolling ball radius) tool before stacking and montage creation.