CD95 ligand (CD95L) immunohistochemistry: a critical study on 12 antibodies

Abstract

In recent years, some studies on the expression of CD95(Fas/ APO-1) ligand (CD95L) in tissues or cells raised concerns about the specificity of the antibodies used. We therefore tested 12 CD95L antibodies for their reliability in immunocyto/ histochemistry by (i) staining CD95L-transfected and control CV-1/EBNA cells and (ii) comparing staining patterns in immunohistochemically labeled tissue sections with the localization of CD95L + cells in in situ hybridization. While G247-4, NOK-1, NOK-2, 4H9, and MIKE-1 stained CD95L-transfected cells and did not significantly bind to controls, G247-4 was the only antibody giving satisfying signals in tissue sections perfectly matching the distribution of CD95L + cells by in situ hybridization. MAb 33, C-20, and N-20 comparably stained both transfected and control cells and showed considerable background or falsely positive staining in sections. MIKE-2, 8B8, A11, and 4A5 did not or only very faintly bind to either cells and, thus, were not tested on sections. We conclude that G247-4 is the only tested antibody that is recommendable for immunohistochemistry. Cell Death and Differentiation (2001) 8, 273 ± 278.

Strategies for immunohistochemical protein localization using antibodies: What did we learn from neurotransmitter transporters in glial cells and neurons

“…This has also been pointed out by multiple investigators (e.g. Strater et al, 2001;Bodei et al, 2009;Jensen et al, 2009;Herber et al, 2004;Grimsey et al, 2008;Kirkpatrick, 2009;Herrera et al, 2013;Bohmer et al, 2014;Baker, 2015;Van Liefferinge et al, 2016).…”

“…These data demonstrate that lack of selectivity appears to be the rule rather than the exception for antibodies against G-protein-coupled and perhaps also other receptors (Michel et al, 2009;Kirkpatrick, 2009). Indeed, there are several reports of poor commercial antibodies to adrenergic receptors (Hamdani and van der Velden, 2009;Jensen et al, 2009) as well as to other proteins such as acetylcholine receptors (Herber et al, 2004;Moser et al, 2007), histones (Egelhofer et al, 2011), CD95 ligand (Strater et al, 2001), angiotensin receptors (Hafko et al, 2013;Herrera et al, 2013;Elliott et al, 2013), dopamine receptors (Bodei et al, 2009), cannabinoid receptors (Snyder et al, 2010;Ashton, 2012;Morozov et al, 2013), histamine receptors (Beermann et al, 2012), P2X receptors (Ashour et al, 2006) and others.…”

Prognostic impact of FAS/CD95/APO-1 in urothelial cancers: decreased expression of Fas is associated with disease progression

“…This discrepancy cannot be fully explained, but it may be due to the use of different clones of antibodies and the large gap in patient number. There is problem in the reliability of antibodies for FasL, and several papers have tested their specificity; many clones including A11 and H11were listed as unreliable, on the other hand, because clone Q-20 we used had not been examined, we cannot give any other evidences besides our own results for the reliability of the antibody, but other clones of Santa Cruz were considered not applicable for IHC (Strater et al, 2001). As for the patients' survival, FasL expression showed no correlation with it.…”

The Contribution of the Fas/FasL Apoptotic Pathway in Ulcer Formation during Leishmania major-Induced Cutaneous Leishmaniasis

“…Deparaffination and rehydration were performed as previously described. 19 Sections were incubated with mouse anti-Fas monoclonals (Dakopatts, Stockholm, Sweden) at 10 g/ml, mouse anti-FasL monoclonals (20 g/ml) (BD, Stockholm, Sweden), or isotype controls (20 g/ml) (Dakopatts) for 15 minutes at room temperature. Streptavidin-avidin enhancement was performed according to the manufacturer's instruction (Dakopatts).…”