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Our Abpromise guarantee covers the use of ab28484 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||Use a concentration of 2 µg/ml. Detects a band of approximately 280 kDa (predicted molecular weight: 156 kDa).|
|ICC/IF||Use at an assay dependent concentration.|
|IHC-P||Use a concentration of 4 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
ab28484 (4µg/ml) staining lubricin in Human liver using an automated system. Using this protocol there is staining of the cytoplasm and nuclei in hepatic arteries and plates of hepatic cells.
Sections were rehydrated and antigen retrieved with buffers EDTA pH 9.0 . Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.
For permeabilization, 0.25% Triton X-100 was included in primary and secondary antibody incubation buffers.
Cells were also stained with FITC-labeled Peanut Agglutinin. PNA binds lubricin and certain other glycoproteins, so some co-localization was expected between the Alexa Fluor 568 signal and the FITC signal.
Unexpected co-localization was noted between the Alexa Fluor 568 staining (indirect lubricin staining) and DAPI (nuclear counterstain) in the cell nuclei.
Primary antibody was diluted in antibody diluent using a Fast Western Blot kit and followed by the optimized HRP reagent. Chemiluminescent detection was performed using SuperSignal West Dura.
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