Development of an optimal protocol for antigen retrieval: a 'test battery' approach exemplified with reference to the staining of retinoblastoma protein (pRB) in formalin-fixed paraffin sections.
Author(s): Shi SR, Cote RJ, Yang C, Chen C, Xu HJ, Benedict WF, Taylor CR
Publication: J Pathol, 1996, Vol. 179, Page 347
PubMed ID: 8774494 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
-
Study Purpose
The purpose of this study was to identify the optimal antigen retrieval solution (Tris HCl or acetate, citrate, phosphate buffers), pH (1, 6, 10), and temperature (90-120 degrees C) for pRB immunohistochemical staining of FFPE normal spleen, small cell lung carcinoma, and bladder carcinoma.
Summary of Findings:
The authors observed optimal and intense pRB immnostaining with the following antigen retrieval conditions: (a) acetate buffer, pH 1, at 100 degrees C for 10 minutes; (b) Tris-HCl buffer, pH 10, at 120 degrees C (autoclave) for 10 minutes or 100 degrees C (microwave) for 1 hour. pRB localization was appropriate although immunostaining was more intense using the above conditions compared to fresh frozen controls. Immunopositive staining was dependent on cancer status. Identical results were reported with monoclonal and polyclonal antibodies.
Biospecimens
Preservative Types
- Formalin
- Frozen
Diagnoses:
- Not specified
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Protein Immunohistochemistry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Snap frozen
Analyte Extraction and Purification Antigen retrieval Acetate buffer
Citrate buffer
Phosphate buffer
Analyte Extraction and Purification Analyte isolation method Monoclonal antibody
Polyclonal antibody
Biospecimen Aliquots and Components pH 1
6
10
Analyte Extraction and Purification Temperature of heat-induced retrieval 90 degrees C (water bath or microwave)
100 degrees C (microwave)
120 degrees C (autoclave)