Confocal microscopy in large insect brains: Zinc-formaldehyde fixation improves synapsin immunostaining and preservation of morphology in whole-mounts

Confocal microscopy enables the analysis of immunofluorescence in whole-mount brains and is therefore widely used in the functional and comparative neuroanatomy of invertebrates. Three difficulties, however, are commonly encountered. First, poor penetration of antibodies after formaldehyde fixation impedes the immunostaining in central neuropile regions. Second, formaldehyde can cause a loss of antigenicity by epitope masking. Third, large brains must be cleared in hydrophobic media, a procedure that may distort morphology. I present a new methodology that overcomes these three problems by using zinc-formaldehyde (ZnFA) for fixation. The success of this technique is demonstrated in the brain of the desert locust and evaluated by comparison with fixation in formaldehyde and immunostaining against synapsin to reveal the regions of synaptic integration throughout the brain. ZnFA fixation markedly increased antibody penetration, prevented synapsin epitope masking, and in the cleared preparation the morphology of the brain was preserved with great fidelity. Possible mechanisms responsible for these improvements are discussed. Successful double labelling for synapsin and serotonin shows that small-molecule antigens are also retained by ZnFA fixation. The methodology should facilitate a range of applications including whole-mount brain stereology and the generation of digital standard brains. It may furthermore facilitate the detection of other protein antigens in large intact specimens such as vertebrate embryos.