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Staining tissue with methacrylate casts for light microscopy
1Department of Pathology, University of Illinois at Chicago 60612-7323, USA.
Abstract:
Scanning electron microscopy (SEM) of methyl methacrylate casts and light microscopy (LM) of tissue are well-established methods for studying the microcirculation. The two are complimentary, but methacrylate is transparent and thus its presence is often not appreciated by LM. Applying histologic stains to sections of tissue embedded in methyl methacrylate would allow the relationships of light microscopic and scanning electron microscopic views of cast vasculature to be better appreciated. We sought to test different stains on cast tissue to find one that would accent the cast. Surgically removed and autopsied human lungs were cast with methacrylate and processed by routine light microscopic methods. They were stained with the hematoxylin and eosin, Masson trichome, elastic--van Gieson, Grocott methenamine silver, Brown-Brennan, and Ziehl-Neelsen methods. The Ziehl-Neelsen procedure stained the methacrylate best, giving it a red color. This procedure also worked well without heating. We conclude that (1) cast methacrylate lung can be processed for routine LM with excellent results; (2) methacrylate stains well with the Ziehl-Neelsen technique; (3) the acid--fast stained cast lung shows capillaries and cells in both normal and diseased lung better than the routine hematoxylin and eosin stain; (4) this technique can be used to assess filling and correlate findings on the same tissue with the two different microscopic methods.
Insights
Histologic stains improve light microscopy of methacrylate casts in human lung microcirculation. The Ziehl-Neelsen stain best accentuates the cast, enhancing visualization of capillaries and cells in both normal and diseased lung tissue.
Area of Science:
- Histology
- Microscopy
- Pulmonary Medicine
Background:
- Scanning electron microscopy (SEM) and light microscopy (LM) are established methods for studying microcirculation.
- Methacrylate casts are used in SEM but are transparent and difficult to appreciate with LM.
- Improved LM visualization of methacrylate casts could enhance correlation between SEM and LM findings.
Purpose of the Study:
- To evaluate different histologic stains for their ability to accentuate methacrylate casts in human lung tissue.
- To determine if staining methacrylate casts improves the appreciation of microvascular structures and cellular detail under LM.
- To assess the utility of stained casts for correlating SEM and LM findings.
Main Methods:
- Human lung tissue (surgically removed and autopsied) was cast with methyl methacrylate.
- Cast lung tissue was processed using routine LM methods.
- Various stains were applied, including hematoxylin and eosin, Masson trichrome, elastic-van Gieson, Grocott methenamine silver, Brown-Brennan, and Ziehl-Neelsen.
Main Results:
- The Ziehl-Neelsen stain effectively stained the methacrylate cast red, with optimal results achieved without heating.
- Acid-fast staining of cast lung tissue provided superior visualization of capillaries and cells in both normal and diseased lungs compared to hematoxylin and eosin.
- Methacrylate casts in lung tissue can be successfully processed for routine LM.
Conclusions:
- The Ziehl-Neelsen technique is highly effective for staining methacrylate casts in lung tissue for LM.
- This method enhances the visualization of microcirculation and cellular details, improving the correlation between SEM and LM.
- Stained methacrylate lung casts offer a valuable tool for assessing microvascular filling and correlating findings from different microscopic techniques.