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Glial Cells in the Optic Lobe - Materials and Methods |
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E. Eule, S. Tix (1), and K.-F. Fischbach
Institut für Biologie III, Schänzlestrasse 1, 79104 Freiburg
i. Br.,
Germany.
(1) New address: California Institute of Technology, Pasadena, California
91125, USA
87 enhancer trap lines isolated in the lab of C. Goodman were kindly given to us by Ch. Klämbt (Köln). They were screened for post embryonic reporter gene expression in glial cell types of the optic lobe. The following six enhancer trap lacZ in-sertion lines fulfilling our criteria were studied in detail: 2-25/CyO; 3-66/TM2; 3-74/TM2; 3-93/TM2; 3- 97/TM2; 3-159/TM2. All lines house the P-vector p(ry+,lacZ) encoding a reporter gene product targeted to the cell nucleus. With the exception of line 3-97, which is an embryonic lethal, all lines are homozygously viable and normal with regard to nervous system structure. In order to adjust the genetic background conditions and to exclude ß-galactosidase dosage effects, all anatomical studies were carried out with heterozygous F1 flies derived from a cross between virgins of the enhancer trap line and WT-Berlin males. The lacZ expressing offspring carries only one copy of the lacZ P- element. Two additional lines T 601; T 1184, isolated in the lab of G.M. Technau, were used. They carry a P-Gaw B-construct that codes for the yeast derived Gal4-transcription factor, the expression of which is under control of Drosophila enhancers (Brand and Perrimon, 1993). When crossed with an UAS-lacZ strain, ß-galactosidase expression is restricted to Gal4 expressing cells in the F1 generation and directed to the cytoplasm.
The flies were first immobilised on ice or etherized before arranging them in groups of up to 5 individuals on a 'collar' normally used for paraffin mass histology (Heisenberg and BÖhl 1978, Jäger and Fischbach 1987). In this approach the flies are attached in a fixed position, facilitating a quick and efficient way of dissection. The collars were cov ered with PBS, and the brains were removed from the head capsules with Dumont Inox 5 forceps. The tissues were immediately transferred into a fixation solution consisting of 2% glutar-dialdehyde (Merck) in PBS. Fixation was performed on ice for 30-45 min before washing 3 x 5 min with PBS. The brains were incubated at 37¡C over night in X-gal staining buffer (Simon et al. 1985) containing 33 µl 8% X-gal (Biomol) in DMSO per ml staining buffer (0.27% ). The tissues were then washed 3 x 5 min in PBS and post fixed in 5% glutardialdehyde in PBS for 1 h at RT prior to either embedding in Glycergel (DAKO) as whole mounts or preparation for semithin sectioning.
After another 3 washes with PBS, brains were dehydrated at RT (10 min in 30% , 50% , 70% , 90% , 2x dried 100% ethanol), incubated 2 x 10 min at RT in xylene, then 1 h at RT in a 1:2 mixture of Epon 812 and xylene and over night at 4¡C in a 1:1 mix of xylene and Epon 812. The first steps involving xylene have to be performed in a closed vial to prevent tissue shrinkage. The next day the xylene was evaporated slowly in the fume hood before transferring the specimen into fresh Epon over night at 4¡C. They were then embedded in fresh Epon at RT and polymerised for 12 h at 42¡C and 24-36 h at 65¡C. Semithin sections of 1.5-2.0 µm were cut with a Nova Ultratom Type 2388 and baked on slides at 65¡C over night. Sections were counterstained with a 1:1 mixture of 0.02% Pyronine G in Aqua dest. (Fluka) or 0.02% methyl violet (Serva) in Aqua dest. and 0.1% Borax (Sigma) for 2 min at 65 ¡C. This mix was prepared immediately before use. We also used a stain of 0.02% methylenblue/ 0.02% toluidine / 0.1% borax in aqua dest. The sections were finally mounted with DePex (Serva).
In order to ensure our classification of cells derived from lacZ positive populations we compared them to osmium fixed material. Osmium fixation alone allows a much better tissue conservation and counterstaining quality. Dissection, Epon embedding and sectioning was done as above. Fixation was done for 2 h on ice with a 0.1 M sodium- cacodylate-buffer containing 2% paraformaldehyde, 2% glutardialdehyde and 0.04% CaCl2 , followed by an-other 2 h at RT in 2% OsO4/ 0.1 M sodium -cacody-late/ 0.04% CaCl2 in aqua dest.
In order to distinguish between neuronal and non-neuronal cells, we applied antibody staining with the strictly neuron specific elav antiserum generated in rats, kindly provided by K. White (Robinow and White 1991). Adult flies were transferred onto collars and embedded in Reichert-Jung medium. Sections of about 10 µm were cut on a Frigocut E (Reichert-Jung) cryotome and transferred to poly-L lysine coated slides, dried at RT for 30 min and fixed for 15 min in 1% paraformaldehyde in PBS at RT. After 3 washes with PBS, the X-gal staining was done over night at 37¡C in a humid chamber with a solution made of X-gal staining buffer (as above), 1% gelatine and 0.2% NaN3. After washing with PBS (3 x 5 min) immunohisto-chemistry was performed in the following way: preincubation for 30 min in 500 µl PBTG per slide (PBS/O.1% Triton X 100/0.1% BSA/3% Goat Serum) at RT, then incubation with 200 µl per slide of elav antibody (diluted 1:100 in PBTG) for 4 h at RT in humid box, 4 x 10 min washes in PBTG. 200 µl of a 1:500 diluted PBGT solution of biotinylated anti rat Ig G (Sigma) antibody was added, and in-cubation for 2 h followed as above. After 4 washes 200 µl of streptavidin coupled Vectastain ABC Kit solution (Vector Labs) was applied for 1 h at 37¡C. Then 4 washes in PBTG were performed before 500 µl of DAB solution (0.01% DAB/ 0.01% H2O2 in PBS) was added to each slide. Brown staining should appear after about 5 min. The reaction was stopped with water when appropriate. Embedding was done in Glycergel (DAKO). Sections were photographed with a Zeiss Axiophot microscope equipped with Normarski optics.
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Poster Sessions
Glial Cells in the Optic Lobe
Materials and Methods
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