Related Experiment Videos
Application of microwave technology to the processing and immunolabeling of plastic-embedded and cryosections
1Pharmacological Sciences, Genentech, Inc., South San Francisco, California 94080, USA.
Abstract:
We have adapted existing microwave irradiation (MWI) protocols and applied them to the processing and immunoelectron microscopy of both plastic-embedded and frozen sections. Rat livers were fixed by rapid MW irradiation in a mild fixation solution. Fixed liver tissue was either cryosectioned or dehydrated and embedded in Spurr's, Unicryl, or LR White resin. Frozen sections and sections of acrylic-embedded tissue were immunolabeled in the MW oven with an anti-catalase antibody, followed by gold labeling. Controls were processed conventionally at room temperature (RT). The use of MWI greatly shortened the fixation, processing, and immunolabeling times without compromising the quality of ultrastructural preservation and the specificity of labeling. The higher immunogold labeling intensity was achieved after a 15-min incubation of primary antibody and gold markers under discontinued MWI at 37C. Quantification of the immunolabeling for catalase indicated a density increase of up to fourfold in the sections immunolabeled in the MW oven over that of samples immunolabeled at RT. These studies define the general conditions of fixation and immunolabeling for both acrylic resin-embedded material and frozen sections.
Insights
Microwave irradiation (MWI) significantly accelerates tissue fixation and immunolabeling for electron microscopy. This method enhances immunogold labeling intensity and preserves ultrastructure, offering a faster alternative to room temperature processing.
Area of Science:
- Electron Microscopy
- Biotechnology
- Cell Biology
Background:
- Conventional methods for tissue processing and immunoelectron microscopy are time-consuming.
- Optimizing fixation and labeling protocols is crucial for high-quality ultrastructural analysis.
Purpose of the Study:
- To adapt and apply microwave irradiation (MWI) protocols for tissue processing and immunoelectron microscopy.
- To evaluate the efficiency and effectiveness of MWI in accelerating fixation, embedding, and immunolabeling procedures.
Main Methods:
- Rat livers were fixed using rapid microwave irradiation (MWI) in a mild solution.
- Fixed tissues were cryosectioned or embedded in Spurr's, Unicryl, or LR White resins.
- Immunolabeling of frozen and resin-embedded sections was performed in a microwave oven using an anti-catalase antibody and gold conjugates.
Main Results:
- MWI significantly reduced fixation, processing, and immunolabeling times without compromising ultrastructural preservation.
- Immunogold labeling intensity increased up to fourfold in MWI-processed sections compared to room temperature controls.
- Optimized MWI protocols achieved higher labeling specificity and intensity within shorter incubation periods.
Conclusions:
- Microwave irradiation is an effective technique for accelerating electron microscopy sample preparation.
- MWI offers a substantial improvement in speed and labeling efficiency for immunoelectron microscopy of embedded tissues and frozen sections.
- The study provides general conditions for MWI-assisted fixation and immunolabeling, applicable to various sample types.