An easy, fast and “low-tech”-equipment-requiring alternative method to optimize immunolabelling conditions for pre-embedding immunogold electron microscopy and to correlate light and electron microscopical immunogold labelling results

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.