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Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry
Published on: December 13, 2013
Protocol for ultrarapid immunostaining of frozen sections
T Richter1, J Nährig, P Komminoth
1Institute of Pathology, Technical University Munich, Germany.
Insights
This study presents a 12-minute immunostaining protocol for frozen sections, ideal for rapid intraoperative diagnosis. The enhanced polymer one-step staining (EPOS) system allows for quick and accurate detection of tumor cells, aiding in surgical decisions.
Area of Science:
- Histopathology
- Immunohistochemistry
- Oncology
Background:
- Intraoperative diagnosis requires rapid and reliable immunostaining of frozen sections.
- Current methods can be time-consuming, limiting their utility during surgery.
Purpose of the Study:
- To establish a rapid immunostaining protocol for intraoperative frozen section diagnosis.
- To evaluate the efficacy of the enhanced polymer one-step staining (EPOS) system for key biomarkers.
Main Methods:
- Utilized the EPOS system with antibodies for leucocyte common antigen (LCA), cytokeratin (CK), and anti-melanoma (MEL).
- Optimized sample fixation using 100% acetone (20 seconds for CK/LCA, 2 minutes for MEL).
- Incubated primary antibodies and developed chromogen reactions with 3,3'diaminobenzidine (DAB) at 37°C.
Main Results:
- Achieved reliable and specific immunostaining with labeling intensity comparable to standard protocols.
- The entire procedure, including fixation and staining, was completed in just 12 minutes.
- Demonstrated successful application for tumor classification and detection of tumor cells in sentinel lymph nodes.
Conclusions:
- The developed 12-minute EPOS protocol enables rapid immunostaining of intraoperative frozen sections.
- This method is valuable for intraoperative diagnosis, tumor classification, and sentinel lymph node analysis.
- The protocol offers a significant time advantage for urgent pathological assessments.
Abstract:
Rapid immunostaining of frozen sections within a tolerable time span would be very helpful for intraoperative diagnosis. A protocol was therefore established using the enhanced polymer one-step staining (EPOS) system (Dako) with antibodies against leucocyte common antigen (LCA), cytokeratin (CK), and anti-melanoma (MEL). Best results with reliable and specific immunostaining and a labelling intensity comparable to standard immunostaining protocols were achieved with fixation of samples in 100% acetone for 20 seconds (CK, LCA) or two minutes (MEL), followed by incubation of the primary antibody and development of the chromogen reaction with 3,3'diaminobenzidine (DAB) for three and five minutes at 37 degrees C, respectively. The total procedure takes only 12 minutes, thus enabling rapid immunostaining on intraoperative frozen sections. Apart from its use in tumour classification, this method is especially useful in detecting tumour cells in sentinel lymph nodes.
