Computational protocol: Circadian pacemaker coupling by multi-peptidergic neurons in the cockroach Leucophaea maderae

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Protocol publication

[…] The preparations were examined with a Leica confocal laser scanning microscope TCS SP2 equipped with an acusto-optical beam splitter for separation of excitation and emitted light and with a variable detection filtering system (spectrophotometer) for the arbitrary selection of spectral intervals of emitted light. Most scans were performed with a Leica HC PL apochromat 20×/0.7 dry lens, but a HCX PL apochromat 40×/1.25 oil immersion lens was used for high resolution scans of neuronal fibers. To exclude crosstalk artifacts, all three channels were scanned sequentially, and the detection ranges were separated as far as possible (FITC: excitation with the 488-nm line of an argon laser, detection between 495 and 530 nm; Cy3: excitation with the 543-nm line of a helium/neon laser, detection between 590 and 610 nm; Alexa 633: excitation with the 633-nm line of a helium/neon laser, detection between 680 and 800 nm). All specimens with recognizable backfill labeling (n = 25) were scanned. We scanned every section in which the right AMe, the anterior and posterior PDFMe, and backfilled neurons of the AMe were present (z-distance of single optical sections: 2 μm). In some specimens, the central termination areas of PDF-ir neurons and PDF-ir commissures were additionally scanned with the 40× lens and a z-resolution of 0.5-1 μm. Data evaluation was performed on a graphics computer workstation.To quantify the results, every soma was identified individually through and between image stacks to prevent counting artifacts resulting from pure counting of neuron profiles. To estimate the average numbers of neurons in certain groups, arithmetic means and standard deviations were calculated. If sections were missing or, more frequently, if the low labeling intensity combined with high background labeling did not allow the unambiguous identification of neurons in groups in which otherwise labeled neurons could always be identified, then the preparations were excluded from the calculation of mean numbers for the respective group. […]

Pipeline specifications

Software tools EMBOSS, AME
Application Laser scanning microscopy
Chemicals FMRFamide