For multiplex staining, we followed the Opal protocol staining method36 for the following markers: CD73 (1:200, Abcam, ab91086) with subsequent visualization using fluorescein Cy3 (1:50); CD163 (1:25, Leica Biosystems, NCL-L-CD163) with visualization accomplished using Cy5 (1:50); and CD68 (1:100, Dako, M0876) with visualization using Cy5.5 (1:50). Nuclei were subsequently visualized with DAPI (1:2000). All of the sections were cover-slipped using Vectashield H-1400 mounting media. For multispectral analysis, a detailed methodology was followed as described previously (Stack et al., 2014). Each of the individually stained sections was utilized to establish the spectral library of fluorophores required for multispectral analysis. The slides were scanned using the Vectra slide scanner (PerkinElmer) under fluorescent conditions. For each marker, the mean fluorescent intensity per case was then determined as a base point from which positive cells could be established. Finally, the co-localization algorithm was used to determine percent of CD68, CD163 and CD73 staining.
Ab91086
Manufactured by Abcam
Sourced in United Kingdom
Ab91086 is an antibody product from Abcam. It is a polyclonal antibody developed in rabbit and purified using protein A. The antibody targets an undisclosed protein.
Automatically generated - may contain errors
Lab products found in correlation
M0876, by Agilent Technologies (1 mentions)
Vectra slide scanner, by PerkinElmer (1 mentions)
Western lightning chemiluminescence reagent plus system, by Cytiva (1 mentions)
A5441, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Ab92547, by Abcam (1 mentions)
E cadherin, by GeneTex (1 mentions)
2 protocols using ab91086
1
Multiplex Immunofluorescence Staining Protocol
2
Western Blot Analysis of CD73 Knockdown
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Whole-cell lysates of CD73-shRNA-treated cells were extracted with 300 μL of radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris, 150 mM sodium chloride (NaCl), 1% NP40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS)) and subjected to Western blotting analysis. The membranes were then incubated with polyclonal antibodies against CD73 (ab91086, 1:1000; Abcam, Cambridge, UK), E-cadherin (GTX124178, 1:5000; Genetex, Irvine, CA, USA), vimentin (ab92547, 1:1000; Abcam, Cambridge, UK), snail (#3879, 1:1000; Cell Signaling, Danvers, MA, USA), and β-actin (A5441, 1:10000; Sigma-Aldrich, St. Louis, Missouri, USA). Horseradish peroxidase-conjugated anti-rabbit secondary antibody was added to detect the primary antibodies, and the blots were developed using a chemiluminescence system (Pierce). All resolved protein bands were developed using the Western Lightning Chemiluminescence Reagent Plus system (Amersham Biosciences). All experiments were repeated at least three times, with similar results. The whole Western Blot figures can be found in the Supplementary Materials File .
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