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The initial immunization was performed with a preparation of full length human recombinant GFAP expressed in bacteria and highly purified. Subsequent boosts were performed with GFAP purified from a Triton X-100 extract of myelin associated material from bovine spinal cord, following an "axonal flotation" procedure (Liem et al.). The GFAP was further purified by centrifugation and ion exchange chromatography in 6m urea on DEAE cellulose.
Our Abpromise guarantee covers the use of ab7260 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-FoFr||1/5000. See Abreview.|
|IHC - Wholemount||Use at an assay dependent concentration.|
|WB||1/50000. Detects a band of approximately 55,48 kDa.
This lower 48kDa band is thought to be a degradation product.
|IHC-P||1/5000. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
Immunohistochemistical detection of GFAP antibody - Astrocyte Marker (ab7260) on formaldehyde-fixed paraffin-embedded monkey brain sections. Antigen retrieval step: Heat mediated. Buffer Used: Citric acid pH6. Permeabilization: None. Blocking step: 1% BSA for 10 mins @ 21°C. Primary antibody incubated at 1/2000 for 2 hours @ 21°C in TBS/BSA/azide. Secondary antibody: anti rabbit IgG Conjugated to biotin (1/200). Marmoset brain: astrocytes are clearly and strongly labelled.
ab7260 at 1/10000 dilution staining Mouse cortical astrocytes by Immunocytochemistry. The cells were permeabilized with Triton/HEPES buffer prior to primary application. The antibody was incubated with the cells for 18 hours and then bound antibody was detected with an Alexa Fluor ® 488 conjugated goat anti-rabbit antibody.
This image is courtesy of an Abreview submited by Charmaine Noonan.
Western blot of whole rat cerebellum homogenate stained with ab7260 at dilution of 1:100,000. A prominent band running with an apparent SDS-PAGE molecular weight of ~55kDa corresponds to rodent GFAP. A lower band at ~48kDa is derived from the GFAP molecule.
ab7260 staining rat brain tissue sections by IHC-P. Sections were fixed in formaldehyde and bocoked with a commercialy available blocking agent prior to incubating with ab7260, diluted 1/5000 for 20 hours at 4°C. A HRP conjugated mouse polyclonal (universal HRP polymer detection) antibody was used as the secondary.
This image is courtesy of an anonymous Abreview
IHC - Wholemount of rat retina tissue labelling GFAP (red) with ab7260. Sample was incubated with primary antibody (1/100) for 18 hours at 4°C. A Phycoerythrin-conjugated goat anti-rabbit IgG monoclonal (1/1000) was used as the secondary antibody.
ab7260 staining rat pup cortical preps by ICC/IF. The preps were grown for 14 days in culture and plated onto coverslips. The preps were acid/alcohol fixed and blocked prior to incubation with ab7260. Bound antibody was detected using an Alexa Fluor ®488 conjugated goat polyclonal antibody. Nuclei were visualised using DAPI.
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