Kaiser Lab P[GAL4] Line Methods

People:

Armstrong, J.D., Yang, M-Y. and Kaiser, K.

Experimental Procedures Used:

Drosophila methods
Drosophila were maintained on a 12h dark : 12h light cycle on standard cornmeal/yeast/agar medium at 23¡C-25¡C and 35%-45% relative humidity. The genetic background of all lines studied was effectively Canton S. P[GAL4] lines were generated as described by Brand and Perrimon (1993). GAL4 is a yeast transcription factor that is functional in Drosophila. Its pattern and timing of expression are dependent upon the genomic context of the inserted P[GAL4] element. GAL4 can be used to drive expression of secondary reporters linked to the GAL4-responsive promoter, UASG. Here, the secondary reporter for GAL4 activity was a second chromosome insertion of UASG-lacZ. There is no detectable lacZ expression in the absence of GAL4.

Cryostat sectioning and Xgal staining

12mm frontal cryostat sections of adult heads were fixed on glass slides for 15 min in phosphate buffered saline (PBS) containing 2% glutaraldehyde. After washing in PBS (2x 15 min), the sections were stained in the dark for 2-4 hours at 37¡C in a moist box. The staining solution was 0.2% Xgal (diluted from an 8% stock solution in DMSO) in pre-warmed FeNaP buffer. FeNaP is 10mM NaH2PO4.H20, 10mM Na2HPO4.2H2O, 150mM NaCl, 1mM MgCl2.6H2O, 3.1mM K4(Fe2+CN)6, 3.1mM K3(Fe3+CN)6, 0.3% Triton X-100, pH 7.4. The staining solution was made fresh each day and kept in the dark. After staining, the sections were washed in PBS for 10 min, dehydrated though graded ethanol, and mounted in glycerol/gelatin (Sigma). Sections were photographed on a Leica Orthoplan microscope using a 25x DIC lens.

Whole-mount immunohistochemistry

Intact adult brains were dissected under PBS, fixed in 4% paraformaldehyde for 30 min, and washed twice for 1 hour in PAT (PBS containing 1% Sigma cold fraction V bovine serum albumin and 1% Triton X-100). They were incubated overnight in 3% normal goat serum (SAPU) containing rabbit polyclonal anti-b-gal antibody (Cappel) diluted 1:2000 in PAT; washed three times in PAT for 1 hour; incubated overnight with secondary antibody (fluorescein-labeled goat anti-rabbit IgG; Vector Labs) diluted 1:250 in PAT; washed twice for 1 hour in PAT, and once for 5 minutes in PBS. All of the above was carried out at room temperature. Stained brains were mounted in VectaShield (Vector).

Confocal microscopy

Intact brains were examined with a Molecular Dynamics Multiprobe laser scanning confocal microscope. Excitation (480nm) and emission (530±15nm) filters were appropriate to the fluorescein-based labels of the secondary antibody. Three dimensional reconstructions were performed using the programme 'ImageSpace 3.1' (Molecular Dynamics). Pseudo colour was added using the programme 'NIH-Image' (National Institutes of Health, Washington).

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