Data Analysis

Measuring Leaf Position by Image Analysis As with the collection of data, the software package MetaMorph is used to analyze the image data. Leaves can be identified in MetaMorph by applying a threshold to the images. The threshold effectively reduces the image from an 8-bit grayscale image to a 1-bit image. Each pixel that was darker than the threshold level in the 8-bit image is set to black all other pixels are white. As the seedlings are relatively darker than the light background...

Setting Up Top Count Monitoring

Bioluminescence rhythms in Drosophila are counted using a TopCount. This instrument has an open (i.e., exposed to room conditions) microtiter plate stacker design. By intercalating empty clear plates between white Optiplates, samples contained in the white Optiplates can be exposed to external light while in the stacker, and then cycled into the light-tight counting chamber to measure bioluminescence. Readers should refer to the TopCount manual for details on setting up a TopCount monitoring...

Top Count Measurement

The Packard TopCount Microplate Scintillation and Luminescence Counter utilizes single photomultiplier tubes to measure the light produced from recombinant luxAB or luc reporter cyanobacteria in counts per second (cps). The advantage of using the TopCount to measure circadian bioluminescence is multifold one can screen hundreds of strains at a time, and the automated counting protocol allows for rhythms to be monitored 24 h a day, for weeks at a time, with little attention from an otherwise...

Description of luxAB luxCDE and luc Vectors

We have constructed a number of vectors that contain the promoterless V. harveyi luxAB genes, which encode the luciferase enzyme, and an antibiotic-resistance marker, flanked by the sequences from one of two identified neutral sites of the cyanobacterial genome (Fig. 1). These neutral sites are regions of the S. elongatus chromosome that can be disrupted without any discernable phenotype (neutral site I, NS1, GenBank accession number U30252 neutral site II, NS2, GenBank accession number...

Problems and Troubleshooting

A particular problem arises when experiments are performed in light-dark LD cycles. During the light portion of an experiment, background counts reach values between 50 and 150 counts per second cps , whereas during the dark portion, background is 10 to 40 cps. This means, if you plot an empty well control well without fly after an experiment, you see a beautiful 24-h pseudorhythm, caused by the light-induced background. Also, FFT-NLLS or any other method will tell you that this is a highly...

Transfection of Luciferase Reporter Plasmid Into PAC2 Zebrafish Cells

We have studied the PAC-2 cell line extensively 6,7,14,15 and so have determined a set of electroporation conditions that provide optimal transfec-tion efficiency. All subsequent protocols employ these conditions. 1. Harvest the cells and prepare them for electroporation exactly as described in Subheading 3.3. 2. Dissolve 35 pg of plasmid DNA containing 25 pg of the luciferase reporter plasmid and 10 pg of the DNA carrier in 50 pL of water and mix with a 500-pL aliquot of resuspended cells see...

Roller Tube Culture

Sterilize all surgical tools and instruments that will come in contact with the slices. Also sterilize with 70 ethanol all surfaces of the tissue chopper or other tissue slicer coming in contact with the animal tissue. 2. Clean the cover slips through a series of solvents xylene 24 h , acetone 24 h , and ethanol 24 h followed by boiling in distilled water, to prevent their sticking together. Then sterilize them with dry heat 200 C for 2 h in a glass Petri dish...

Fluorescent ISH Using DIGLabeled Probes

For this ISH method, the whole procedure is carried out on free-floating sections obtained after perfusion fixation, postfixation, sucrose embedding, and cryosectioning of the brain see Note 12 . 1. Anesthetize and perfuse with heparin-PBS followed by 4 PFA using a regular perfusion protocol. 2. Decapitate and remove brain carefully. 3. Postfix overnight 16-24 h in 4 PFA at 4 C. 1. Drain PFA from the vessel containing the brain and fill it with 20 sucrose. 2. Store at 4 C for 48 h this...

Tissue Preparation and Sectioning

The SCN of the hypothalamus is a bilateral structure localized under both sides of the third ventricle, above the optic chiasm. Because of its position inside the brain, the best way for fixing this tissue is by transcardial perfusion. The following fixation protocol employes 4 buffered paraformaldehyde it has been performed successfully with several commercially available antibodies. Alternative fixative solutions are described in Heading 4 see Note 2 . 1. Open skin of thorax and abdomen of...

Notes

Louis, MO, cat. no. D-5758 per liter of distilled water to treat DEPC is toxic and should be used only under a fume hood . Stir with a magnetic bar until in solution. Let sit overnight at room temperature. Boil for approx 2 h in the fume hood, and autoclave. Although some protocols prepare RNase-free water only by autoclaving water treated overnight with DEPC, boiling for 2 h before autoclaving assures that all DEPC is degraded. Autoclaved DEPC water can be...

Dissection and Fixation of Whole Mount Brains

Newly bought forceps are usually not fine enough for good preparations. They need to be sharpened with the help of sandpaper under a dissecting microscope Fig. 1A . The sharpened forceps should then be used only for the dissections use other forceps to grasp flies, etc. . It is also necessary to resharpen the forceps from time to time after regular use. Dissection is preferentially performed in a black block dish filled with PBS. The black background helps to...