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Updated: Aug 21, 2026
A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections
Published on: March 30, 2020
The influence of immunohistochemistry on mRNA recovery from microdissected frozen and formalin-fixed,
Lise Mette Gjerdrum1, Helene N Abrahamsen, Berta Villegas
1Institute of Pathology, Aahus University Hospital, Aarhus, Denmark. lmgje@mail.dk
Abstract:
Laser-assisted microdissection (LAM) is now widely used to obtain specific cell populations from heterogeneous tissues. A major disadvantage of LAM is poor tissue morphology during microscopy, in part because coverslips are not used. Immunohistochemical labeling can improve identification of target cells but may affect the subsequent analysis of the microdissected tissue. We studied the effect of immunohistochemistry (IHC) on mRNA recovery from labeled cells after microdissection from both frozen and formalin-fixed, paraffin-embedded (FFPE) sections, using Melan-A and Ki-67 staining in lymph nodes with metastatic melanoma as a model. We developed rapid protocols for immunostaining in an attempt to limit loss of mRNA during procedures. A sensitive real-time quantitative reverse transcription-PCR was used to measure mRNA. We found a marked decrease in the mRNA yield from 500 microdissected cells from frozen and paraffin sections after immunostaining for both markers. Recovery of mRNA decreased by up to 89%, comparing the immunostained with the routinely stained sections. Interestingly, the ratio between mRNA for the two markers was similar in all stains, indicating that immunostained sections may be used for mRNA analysis. We also investigated the effect of storing membrane-mounted sections for microdissection under different conditions. Slides mounted with paraffin sections could be stored at room temperature for up to 90 days with no significant decrease in mRNA recovery.
Insights
Immunohistochemistry (IHC) significantly reduces mRNA recovery after laser-assisted microdissection (LAM), decreasing yield by up to 89%. However, relative mRNA marker ratios remain consistent, suggesting IHC-labeled cells are still viable for molecular analysis.
Area of Science:
- Molecular Biology
- Histology
- Cancer Research
Background:
- Laser-assisted microdissection (LAM) isolates specific cells but often yields poor tissue morphology.
- Immunohistochemistry (IHC) aids cell identification but may impact downstream molecular analysis.
- Formalin-fixed, paraffin-embedded (FFPE) tissues are common in pathology but can pose challenges for molecular studies.
Purpose of the Study:
- To investigate the impact of immunohistochemistry (IHC) on mRNA recovery following laser-assisted microdissection (LAM).
- To evaluate the effect of rapid IHC protocols on mRNA yield from frozen and FFPE tissue sections.
- To assess the feasibility of using IHC-labeled cells for subsequent mRNA analysis.
Main Methods:
- Laser-assisted microdissection (LAM) was performed on lymph node sections with metastatic melanoma.
- Immunohistochemistry (IHC) for Melan-A and Ki-67 was applied using rapid protocols.
- Real-time quantitative reverse transcription-PCR (RT-qPCR) was used to measure mRNA recovery.
- Storage conditions for membrane-mounted sections were evaluated.
Main Results:
- IHC staining led to a significant decrease in mRNA yield, with recovery reduced by up to 89% compared to unstained sections.
- Despite the reduction in yield, the relative ratios of target mRNA markers remained consistent across different staining conditions.
- Paraffin-embedded sections stored at room temperature for up to 90 days showed no significant loss in mRNA recovery.
Conclusions:
- Immunohistochemistry significantly impacts mRNA yield after laser-assisted microdissection.
- The consistent ratio of mRNA markers suggests that IHC-labeled cells can still be used for relative gene expression analysis.
- Optimized storage protocols for FFPE sections support long-term sample preservation for microdissection and molecular analysis.
