Selection of buffer pH by the isoelectric point of the antigen for the efficient heat-induced epitope retrieval: re-appraisal for nuclear protein pathobiology.
Epitope retrieval (ER) using heating causes a dramatic improvement in the sensitivity of immunohistochemistry for formalin-fixed paraffin-embedded (FFPE) tissue sections. Here, the relationship between the pH of the retrieval buffer used for heat-induced epitope retrieval (HIER) and the isoelectric points (pI) of the antigen recognized by antibodies against nuclear proteins (mainly human pituitary transcription factors in this study) was investigated using FFPE tissue sections. A universal buffer, with a buffering capacity over a wide pH range from 2.0 to 12.0, was used for HIER. We found that the intensity of staining for most nuclear proteins after HIER depended simply on the pH of the buffer. Importantly, for efficient HIER, antigens with acidic pI required basic pH buffer conditions, while antigens with alkaline pI required acidic conditions. This implies that the electrostatic charge of the antigens contributed significantly to the efficiency of HIER. We conclude that appropriate selection of the pH of the buffer based on the pI of the individual antigens is of great importance for efficient ER. It is concluded that the mechanism of HEIR may, therefore, depend to a large extent on the pI of the antigen under investigation.
Kajiya
Hanako
H
Department of Pathology, Tokai University School of Medicine, 143 Shimokasuya, Boseidai Isehara, Kanagawa, 259-1193, Japan. hanakokj@is.icc.u-tokai.ac.jp
Takekoshi
Susumu
S
Takei
Mao
M
Egashira
Noboru
N
Miyakoshi
Takashi
T
Serizawa
Akihito
A
Teramoto
Akira
A
Osamura
Robert Y
RY
D000906:Antibodies; D000941:Antigens; D002021:Buffers; D000939:Epitopes / Q000032:analysis*; D005557:Formaldehyde; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry / Q000379:methods* / Q000592:standards; D007526:Isoelectric Point; D009687:Nuclear Proteins / Q000032:analysis / Q000276:immunology; D016612:Paraffin Embedding; D016707:Tissue Fixation
D000906:Antibodies; D000941:Antigens; D002021:Buffers; D000939:Epitopes; D009687:Nuclear Proteins; D005557:Formaldehyde