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Updated: Mar 7, 2026
High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses
Published on: February 18, 2016
An easy, fast and "low-tech"-equipment-requiring alternative method to optimize immunolabelling conditions for
Shweta Suiwal1, Gabriele Kiefer1, Frank Schmitz1
1Saarland University, Department of Neuroanatomy, Institute of Anatomy and Cell Biology, Kirrbergerstrasse, 66421 Homburg/Saar, Germany.
Insights
This study introduces a low-tech, pre-embedding immunolabelling method for correlating light microscopy (LM) and electron microscopy (EM) data. This accessible technique enables detailed ultrastructural analysis of the same biological samples at both microscopic levels.
Area of Science:
- Biomedical Research
- Microscopy Techniques
- Cell Biology
Background:
- Correlating light microscopy (LM) and electron microscopy (EM) data is crucial but often hindered by specialized equipment and complex protocols.
- Existing methods require expensive, inaccessible technology, limiting their widespread use in research labs.
Purpose of the Study:
- To present an accessible, low-tech pre-embedding immunolabelling approach for correlating LM and EM data.
- To demonstrate the method's utility using immunolabelled synaptic ribbons in retinal rod photoreceptor synapses.
Main Methods:
- A cryostat section-based, pre-embedding immunolabelling procedure using commonly available equipment.
- Optimization of immunolabelling at the LM level before ultrastructural analysis via ultrathin sectioning.
- A detergent-free protocol suitable for weakly fixed cryostat sections.
Main Results:
- Successful correlation of LM immunolabelling with EM data on the same sample.
- Enables sequential analysis of individual photoreceptor synapses at both LM and EM levels.
- The method is adaptable for higher resolution EM analyses with minor modifications.
Conclusions:
- The described approach offers an easy and affordable way to combine LM and EM immunolabelling.
- Facilitates detailed ultrastructural investigation of biological samples, particularly for antibodies requiring gentle fixation.
- Enhances the accessibility of correlative light and electron microscopy in biomedical research.
Abstract:
Correlating light microscopic immunolabelling results with electron microscopic data is of great interest in many fields of biomedical research but typically requires very specialized, expensive equipment and complex procedures which are not available in most labs. In this technical study, we describe an easy and "low-tech"-equipment-requiring pre-embedding immunolabelling approach that allows correlation of light microscopical immunolabelling results with electron microscopic (EM) data as demonstrated by the example of immunolabelled synaptic ribbons from retinal rod photoreceptor synapses. This pre-embedding approach does not require specialized embedding devices but only commonly available equipment. The cryostat section-based procedure allows optimization of the pre-embedding immunolabelling conditions at the less laborious and time-consuming light microscopic (LM) level before the ultrastructural analyses of the immunolabelled structures can be performed on the same sample after ultrathin sectioning without further modification. The same photoreceptor synapse that has been first studied at the light microscopic level can be subsequently analyzed with this approach at the electron microscopic level at individual ultrathin sections or serial ultrathin sections from individual, identical synapses. Higher resolution EM analyses of the immunolabelled synapses can be performed with only minor modifications of the combined LM/EM procedure. The detergent-free procedure is applicable even for weakly fixed cryostat sections which is a relevant aspect for many antibodies that do not work with more strongly fixed biological samples.
