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
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Abbreviations
- CD95L:
-
CD95 ligand
- FACS:
-
fluorescence-assisted cell scan
- kDa:
-
kilo Dalton
- mAb:
-
monoclonal antibody
- mRNA:
-
message ribonucleic acid
- PBS:
-
phosphate buffered saline
- RT–PCR:
-
reverse transcriptase-polymerase chain reaction
- TRAIL:
-
tumor necrosis factor-related apoptosis-inducing ligand
References
Suda T, Takahashi T, Golstein P, Nagata S . 1993 Molecular cloning and expression of the Fas ligand, a novel member of the Tumor Necrosis Factor family Cell 75: 1169–1178
Takahashi T, Tanaka M, Inazawa J, Abe T, Suda T, Nagata S . 1994 Human Fas ligand: gene structure, chromosomal location and species specificity Int. Immunol. 10: 1567–1574
van-Parijs L, Abbas -AK . 1996 Role of Fas-mediated cell death in the regulation of immune responses Curr. Opin. Immunol. 8: 355–361
Griffith TS, Ferguson TA . 1997 The role of FasL-induced apoptosis in immune privilege Immunol. Today 18: 240–244
O'Connell J, Bennett MW, O'Sullivan GC, Collins JK, Shanahan F . 1999 The Fas counterattack: cancer as a site of immune privilege Immunol. Today 20: 46–52
Stokes TA, Rymaszewski M, Arscott PL, Wang SH, Bretz JD, Bartron J, Baker Jr JR . 1998 Constitutive expression of FasL in thyrocytes Science 279: 2015
Fiedler P, Schaetzlien CE, Eibel H . 1998 Constitutive expression of FasL in thyrocytes Science 279: 2015
Smith D, Sieg S, Kaplan D . 1998 Aberrant detection of cell surface Fas ligand with anti-peptide antibodies J. Immunol. 160: 4159–4160
Fiedler P, Eibel H . 2000 Antibody mAb33 from Transduction Laboratories detects human CD95L in ELISA but not in immunoblots Cell Death Differ. 7: 126–128
Baker Jr JR, Bretz JD . 2000 Specificity questions concerning the clone 33 anti-fas ligand antibody Cell Death Differ. 7: 8–9
Sträter J, Mariani SM, Walczak H, Rücker FG, Leithäuser F, Krammer PH, Möller P . 1999 CD95 ligand (CD95L) in normal human lymphoid tissues: a subset of plasma cells are prominent producers of CD95L Am. J. Pathol. 154: 193–201
Giordano C, Stassi G, DeMaria R, Toldaro M, Richiusa P, Papoff G, Ruberti G, Bagnasco M, Testi R, Galluzzo A . 1997 Potential involvement of Fas and its ligand in the pathogenesis of Hashimoto's thyroiditis Science 275: 960–963
Herr I, Posovsky C, Bohler T, Debatin K-M . 2000 mAb 33 from Transduction Laboratories specifically binds to human CD95-L Cell Death Differ. 7: 129–130
Walczak H, Degli-Esposti MA, Johnson RS, Smolak PJ, Waugh JY, Boiani N, Timour MS, Gerhart MJ, Schooley KA, Smith CA, Goodwin RG, Rauch CT . 1997 TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL EMBO J. 16: 5386–5397
Younes M, Schwartz MR, Ertan A, Finnie D, Younes A . 2000 Fas ligand expression in esophageal carcinomas and their lymph node metastases Cancer 88: 524–528
Bennett MW, O'Connell J, O'Sullivan GC, Brady C, Roche D, Collins JK, Shanahan F . 1998 The Fas counterattack in vivo: apoptotic depletion of tumor-infiltrating lymphocytes associated with Fas ligand expression by human esophageal carcinomas J. Immunol. 160: 5669–5675
Bennett MW, O'Connell J, O'Sullivan GC, Roche D, Brady C, Kelly J, Collins JK, Shanahan F . 1999 Expression of Fas ligand by human gastric adenocarcinomas: a potential mechanism of immune escape in stomach cancer Gut 44: 156–162
Möller P, Walczak H, Riedl S, Sträter J, Krammer PH . 1996 Paneth cells express high levels of CD95 ligand transcripts. A unique property among gastrointestinal epithelia Am. J. Pathol. 149: 9–13
Acknowledgements
The authors thank S Westenfelder, K Pruy, and H Stahl for excellent technical assistance. The study was supported by grants from the Deutsche Forschungsgemeinschaft (D.0687 STR 543/1-1; SFB518/A6). H Walczak was supported by a BioFuture grant from the Bundesministerium für Bildung und Forschung (BMBF).
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Sträter, J., Walczak, H., Hasel, C. et al. CD95 ligand (CD95L) immunohistochemistry: a critical study on 12 antibodies. Cell Death Differ 8, 273–278 (2001). https://doi.org/10.1038/sj.cdd.4400813
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DOI: https://doi.org/10.1038/sj.cdd.4400813
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