Journal article
New immunohistochemical "sandwich" staining method for mdr1 P-glycoprotein detection with JSB-1 monoclonal antibody in formalin-fixed, paraffin-embedded human tissues
The American journal of pathology, Vol.144(2), pp.227-236
02/01/1994
PMCID: PMC1887155
PMID: 7508682
Abstract
We have developed a new immunoperoxidase "sandwich" staining method for amplified detection of P-glycoprotein (Pgp) that is suitable for use on formalin-fixed, paraffin-embedded (conventional) tissue sections. This was accomplished by substantially changing the procedure described by Chan (1988) so as to increase specific staining intensity and to decrease nonspecific background staining. To determine the most appropriate primary antibody for the assay, we compared the immunoreactivity of JSB-1, C494, and C219 monoclonal antibodies recognizing internal epitopes of Pgp, and MRK16 and 4E3 monoclonal antibodies recognizing external epitopes of Pgp. Paraffin sections of Pgp-positive normal human tissues (adrenal, liver, kidney, and brain), of renal tumors, and of cell pellets of sensitive and multidrug resistant human tumor cell lines (MCF-7, KB) were used for comparisons. Immunostaining was excellent with JSB-1, moderate with C494, and very weak with C219. MRK16 and 4E3 showed no reaction. Nonspecific background staining was reduced by 1) omitting immunoglobulin G from secondary antibodies; 2) decreasing the concentration of peroxidase-antiperoxidase complex; and 3) utilizing casein solution for blocking and washing. Pretreatment of sections before immunostaining was also simplified. Using JSB-1, the threshold for detection of elevated Pgp corresponded to less than two-fold relative resistance to doxorubicin. Applying this method, we found two of 26 non-small cell lung cancers were positive for Pgp, consistent with previous results of others using frozen sections. This new immunoperoxidase sandwich staining method using JSB-1 now allows reliable Pgp detection in sections of formalin-fixed, paraffin-embedded (archived) surgical specimens and small biopsy materials commonly used for diagnostic purposes.
Details
- Title: Subtitle
- New immunohistochemical "sandwich" staining method for mdr1 P-glycoprotein detection with JSB-1 monoclonal antibody in formalin-fixed, paraffin-embedded human tissues
- Creators
- K. Tóth - Roswell Park Cancer InstituteM. M. Vaughan - Roswell Park Cancer InstituteH. K. Slocum - Roswell Park Cancer InstituteM. A. Arredondo - Roswell Park Cancer InstituteH. Takita - Roswell Park Cancer InstituteR. M. Baker - Roswell Park Cancer InstituteY. M. Rustum - Roswell Park Cancer Institute
- Resource Type
- Journal article
- Publication Details
- The American journal of pathology, Vol.144(2), pp.227-236
- PMID
- 7508682
- PMCID
- PMC1887155
- ISSN
- 0002-9440
- eISSN
- 1525-2191
- Language
- English
- Date published
- 02/01/1994
- Academic Unit
- Hematology, Oncology, and Blood & Marrow Transplantation; Internal Medicine
- Record Identifier
- 9984359762102771
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