Brief freezing steps lead to robust immunofluorescence in the Drosophila nervous system

Abstract

Drosophila melanogaster possesses a complex nervous system, regulating sophisticated behavioral outputs, that serves as a powerful model for dissecting molecular mechanisms underlying neuronal function and neurodegenerative disease. Immunofluorescence techniques provide a way to visualize the spatiotemporal organization of these networks, permitting observation of their development, functional location, remodeling and, eventually, degradation. However, basic immunostaining techniques do not always result in efficient antibody penetration through the brain, and supplemental techniques to enhance permeability can compromise structural integrity, altering spatial organization. Here, slow freezing of brains is shown to facilitate antibody permeability without loss of antibody specificity or brain integrity. To demonstrate the advantages of this freezing technique, the results of two commonly used permeation methods - detergent-based and partial proteolytic digestion - are compared.

Keywords: Drosophila; Drosophila melanogaster; IF; Per; Period; antibody; brain; circadian; flies; fluorescence; freezing; imaging; immunofluorescence; microscopy; nervous system; neural circuit; phalloidin; sleep; staining; wake.

Figure 1.. Comparison of four immunofluorescence techniques reveals quantitative and qualitative differences in detection of circadian clock neurons.

Oregon-R third instar larval brains were dissected and stained using the indicated immunofluorescence techniques. Anti-Per was used to specifically detect circadian clock neurons. Maximum projections from three representative brains are shown from each technique. Scale bars: 20 μm. Microscope settings were kept constant across techniques. Voxels for each lobe were quantified and the data is shown. A t-test was used to determine whether total signal detected by each technique was significantly different from the basic protocol. Diagrammed are locations of per/tim-expressing cells and their projections in the larval brain, reproduced from Helfrich-Forster [9].

Figure 2.. Detail of third instar larval brains focusing on anti-Per immunofluorescence in axons shows quantitative and qualitative differences.

Oregon-R third instar larval brains were dissected and stained using the techniques indicated. Anti-Per was used to stain clock neurons. Brains were imaged and analyzed as above, focusing on the axons displayed. Maximum projections from three representative brains from each technique are shown. Scale bars: 20 μm. A dotted-outline on the diagram, described above, shows the location of enlargement.

Figure 3.. Comparison of immunofluorescence techniques reveals equal staining of F-actin, but reveals distortion of lobe shape upon enzyme treatment.

Oregon-R third instar larval brains were dissected and stained using the indicated techniques. Phalloidin was used to stain F-actin and visualize the cytoskeleton. Brains were imaged and analyzed as above. Maximum projections from three representative brains from each technique are shown. Scale bars: 10 μm.

Figure 4.. Visualizing DNA/nuclei reveals differences in maintenance of brain-region organization and overall shape between immunofluorescence techniques.

Oregon-R third instar larval brains were dissected and stained using the indicated techniques. DAPI was used to stain DNA/nuclei. Brains were imaged as above. Maximum projections from three representative brains from each technique are shown. Scale bars: 20 μm.

Figure 5.. In adult Drosophila brains, freezing leads to qualitative but not quantitative differences in clock neuron staining.

Immunostaining of Oregon-R adult brains using an anti-Per antibody to reveal circadian clock neurons. Brains were imaged and analyzed as above. Maximum projections from three representative brains from each technique are shown. Scale bars: 20 μm. Diagrammed are the locations of per/tim-expressing cells and their projections in the adult brain, reproduced from Helfrich-Forster [9]. DN: Dorsal Neurons; H-B: Photoreceptor cells of the Hofbauer-Buchner eyelets; LN: Lateral neurons (l: large; s: small; d: dorsal); PL_tim: Posterior lateral brain cells expressing tim; R: photoreceptor nuclei (there are two groups 1–6 and 7/8).

Figure 6.. In adult brains, detection of F-actin is increased by including a freezing step.

Oregon-R adult brains were dissected and stained using Phalloidin to stain F-actin. Brains were imaged and analyzed as above. Maximum projections from three representative brains from each technique are shown. Scale bars: 20 μm.

Figure 7.. Imaging of DNA/nuclei in adult brains reveals comparable maintenance of overall morphology after freezing.

Oregon-R adult brains were dissected and stained using each of the three techniques detailed in the protocol. DAPI was used to stain DNA/nuclei. Brains were imaged as above. Maximum projections from three representative brains from each technique are shown. Scale bars: 20 μm.