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A comparative study on tissue processing procedures for the immunohistochemical investigation of oral mucosal
A W Barrett1, A D Beynon, D J Reid
1Department of Oral Biology, University of Newcastle-upon-Tyne, UK.
Insights
For accurate oral mucosal Langerhans cell counts, frozen tissue with anti-CD1a antibody is optimal. Wax-embedded tissue may yield unreliable results for immunohistochemistry studies.
Area of Science:
- Immunohistochemistry
- Oral Mucosal Immunology
- Cell Biology
Background:
- Langerhans cells are crucial immune cells in the oral mucosa.
- Accurate quantification of these cells is vital for understanding oral mucosal immunity and disease.
- Tissue processing methods can significantly impact immunohistochemical results.
Purpose of the Study:
- To compare wax-embedded versus frozen tissue for quantitative immunohistochemistry of oral mucosal Langerhans cells.
- To determine the optimal antibody and tissue preparation method for reliable Langerhans cell counting.
- To assess the impact of tissue processing on antigenicity and cell morphology.
Main Methods:
- Immunoperoxidase technique utilized on both frozen and wax-embedded oral mucosal tissues.
- Comparison of anti-CD1a, -HLADR, and -S100 antibodies for Langerhans cell detection.
- Evaluation of factors including fixative, antibody dilution, and trypsinisation on cell counts.
Main Results:
- Anti-HLADR detected Langerhans cells in both tissue types, but counts were higher in frozen sections.
- Anti-CD1a antibody consistently yielded higher counts in frozen tissue compared to wax-embedded.
- Tissue processing unpredictably affected HLADR antigenicity, while CD1a counts were more reliable.
Conclusions:
- The use of anti-CD1a antibody on frozen oral mucosal tissue is the preferred method for quantitative immunohistochemistry.
- Wax-embedded tissue processing may lead to unreliable quantitative data for oral mucosal Langerhans cells.
- Standardization of tissue preparation is critical for reproducible immunohistochemical studies.
Abstract:
An immunoperoxidase technique was used to compare wax-embedded tissue with frozen tissue for quantitative immunohistochemistry of oral mucosal Langerhans cells. Initial experiments using anti-CD1a, -HLADR and -S100 antisera showed that phenotype, fixative, antibody dilution and trypsinisation of the tissue section significantly affected Langerhans cell counts. Only the anti-HLADR antibody detected Langerhans cells in both frozen and wax-embedded sections. Some 38% of S100-positive dendritic cells were situated in the stratum basale, and 41-84% of these contained melanin as determined by double-labelling. Sections from 39 volunteers were then reacted with the anti-CD1a and -HLADR antibodies. The morphology of Langerhans cells was more dendritic in frozen sections, and the mean HLADR-positive Langerhans cells count in frozen sections was significantly higher than that in wax-embedded sections from the same individual. The intra-individual ratio of counts between frozen and wax-embedded sections was variable; hence, the apparent loss of HLADR antigenicity as a result of tissue processing was unpredictable. Counts of CD1a-positive Langerhans cells were consistently higher. We conclude that the use of anti-CD1a antibody on frozen tissue is the optimum method for quantitative studies of oral mucosal Langerhans cells, and that such studies performed on wax-embedded tissue may be unreliable.