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Rapid microwave fixation of human tissues for light microscopic immunoperoxidase identification of diagnostically
G R Login1, S J Schnitt, A M Dvorak
1Department of Pathology, Beth Israel Hospital, Boston, Massachusetts.
Abstract:
Microwave (MW) energy permits rapid tissue fixation for light and electron microscopy but its effects on antigen preservation have not been fully evaluated. We, therefore, fixed three samples of human skin, uterus, and cervix, and two samples of human colon and breast by MW irradiation (5 to 8 seconds) during simultaneous immersion in a dilute aldehyde mixture (2% formaldehyde and 0.05% glutaraldehyde). For comparison, similar portions of each specimen were fixed in formalin. Specimens were processed routinely and embedded in paraffin for light microscopy. Sections from each specimen were stained with hematoxylin and eosin and, by immunoperoxidase techniques, for epithelial membrane antigen, leukocyte common antigen, S-100 keratin, carcinoembryonic antigen, and factor VIII-related antigen, the latter three with and without preliminary trypsinization. Colon sections were also stained for chromogranin. In all cases, light microscopic morphology was comparable for tissues fixed by the MW method and formalin-fixed specimens, as was immunostaining for epithelial membrane antigen, leukocyte common antigen, S-100 protein, and chromogranin. Formalin-fixed tissues required trypsinization for optimal detection of keratin, carcinoembryonic antigen, and factor VIII-related antigen. In contrast, trypsin-pretreatment was not necessary to demonstrate these antigens in MW-fixed specimens and, in fact, resulted in tissue digestion. We conclude that this MW fixation method provides a means for rapidly fixing tissues for immunoperoxidase staining while preserving excellent light microscopic morphology.
Insights
Microwave (MW) fixation rapidly preserves tissue morphology and antigens for immunoperoxidase staining. This method eliminates the need for trypsinization, unlike traditional formalin fixation, for optimal antigen detection.
Area of Science:
- Histopathology
- Biotechnology
- Immunohistochemistry
Background:
- Microwave (MW) energy offers rapid tissue fixation for microscopy.
- The impact of MW fixation on antigen preservation requires thorough evaluation.
Purpose of the Study:
- To assess the efficacy of MW fixation combined with aldehyde mixture for antigen preservation.
- To compare MW fixation with traditional formalin fixation for light microscopy and immunoperoxidase staining.
Main Methods:
- Human tissue samples (skin, uterus, cervix, colon, breast) were fixed using MW irradiation with aldehydes or formalin.
- Tissues were embedded in paraffin and stained using hematoxylin and eosin.
- Immunoperoxidase techniques were employed to detect various antigens, with and without trypsinization.
Main Results:
- Light microscopic morphology was comparable between MW-fixed and formalin-fixed tissues.
- Immunostaining for several antigens (epithelial membrane antigen, leukocyte common antigen, S-100 protein, chromogranin) was similar in both fixation methods.
- MW-fixed tissues showed optimal detection of keratin, carcinoembryonic antigen, and factor VIII-related antigen without trypsinization, which degraded formalin-fixed tissues.
Conclusions:
- MW fixation is a rapid method for preparing tissues for immunoperoxidase staining.
- This technique preserves excellent light microscopic morphology and enhances antigen detection.
- MW fixation eliminates the need for trypsinization, simplifying the immunohistochemistry workflow.