Egg chamber

Xue F, Cooley L. kelch encodes a component of intercellular bridges in Drosophila egg chambers. Cell 1993 72(5) 681-693. 

Protocol III.C.2 is also a mass preparative procedure, but differs from Protocol III.C.l in that it fixes the egg chambers immediately as they are released from... 

Both of these mass preparative procedures take advantage of the fact that egg chambers are among the densest of fly tissues and can therefore be separated from contaminants by repeated settling and washing. 

Protocol III.C.3 is the method of choice when the number of female flies is limiting, as it often is when working with special stocks such as those carrying meiotic mutations. One can easily obtain enough egg chambers for a hybridization experiment from 6-12 females by this dissection method, and the different stages are more evenly represented, but it is more laborious. 

The three methods give essentially equivalent results. Morphological preservation is subtly better with the latter two protocols, but Protocol III.C.l is the least noxious to carry out. Younger egg chambers tend to fix more reproducibly and to hybridize more intensely (Fig. 3, see color plate). 

Resuspend egg chambers in 100 ml MRS and pour through the fine sieve into another 250-ml beaker. This sieve separates older (stages 11-14) from younger egg chambers. Rinse the retained, older egg chambers into 150-ml beaker. Allow each batch of egg chambers to settle, then aspirate most... 

Allow to settle, remove supernatant, and replace with 10 ml fixative. For older egg chambers, use 8% formadehyde in 1 x cacodylate buffer, above, prewarmed to 37 C. For younger egg chambers lower the formaldehyde concentration to 5%. 

Egg Chamber Mass Isolation with Instantaneous Formaldehyde Fixation... 

Allow to settle aspirate supernatant add 200 ml 2x SSC and pour egg chambers through the second sieve (280-pim mesh) into a fresh 200-ml beaker. 

Allow to settle resuspend in 100 ml 2X SSC. If desired, the egg chambers can be further sieved at this stage to separate older from younger stages. 

Aspirate most of the solution, and pour the egg chambers into 15-ml conical tubes (the detergent will prevent them from sticking even without BSA treatment). Use for hybridization. 

At the end of the fixation period, use forceps to transfer the egg chambers to a large drop of 2x SSCT. Complete the separation of the tissue into individual ovarioles (this is the tedious part). 

This protocol has been used successfully for Drosophila embryos and egg chambers (Dernburg et ai, 1996a-c Marshall et ai, 1996) and also for maize meiocyte columns (A. F. Dernburg and J. C. Fung, unpublished). 

Notes Throughout this protocol the samples are referred to as "embryos, but substitute egg chambers or other tissue as appropriate. Washing is carried out by allowing the embryos to settle to the bottom of the tube, aspirating most... 

Cellector sieves for Drosophila embryo and egg chamber preparation catalog if 1985-ifffffifff ( various numbers for pans, screens of different mesh, keys for changing screens, or complete Cellector kits) contact company for international distributors]... 

Stage 14 Drosophila egg chambers (hereafter referred to as oocytes) are arrested in first meiotic metaphase. A cell-free extract of these oocytes catalyzes apparent disassembly of purified Drosophila nuclei (purified from either embryos or tissue culture cells) as well as of nuclear lamin polymers formed in vitro from isolated interphase lamins (Maus et al., 1995). Biochemically, the oocyte extract catalyzes lamin solubilization and phosphorylation as well as characteristic changes in one- and two-dimensional gel mobility. Cell-free nuclear lamina disassembly is ATP dependent and addition of calcium to extracts blocks disassembly as judged both morphologically and biochemically. 

Jacobs-Lorena, M., and Crippa, M. (1977). Mass fractionation of Drosophila egg chambers. Dev. Biol. 57,385-392. 

Urban, P.L., Chang, C.H., Wu, J.T. and Chen, Y.C. (2011) Microscale MALDl imaging of outer-layer lipids in intact egg chambers from Drosophila melanogaster. Anal. Chem. 83,3918-3925. 

J. Sen, J. S. Goltz, L. Stevens, D. Stein, Spatially restricted expression of pipe in the Drosophila egg chamber defines embryonic dorsal-ventral polarity. Cell 1998 95, 471-81. 

Protocol 2.1 Restriction Enyzme Fragmentation of Probe DNA, 32 Protocol 2.2 Eabelinc with Terminal Deoxynucleotidyl Transferase, 33 Protocol 2.3 Microdissection of Polyeene Chromosomes for DOP-PCR, 34 Protocol 2.4 Primary DOP-PCR Amplification, 36 Protocol 2.5 Secondary DOP-PCR Amplification, 39 FIXATION METHODS, 41 Embryos, 41 Egg Chambers, 42 Other Tissues, 42... 

Protocol 2.6 Manual Dissection and Fixation of Egg Chambers, 43 Protocol 2.7 Formaldehyde Fixation onto Slides for Whole-mount FISH, 45 HYBRIDIZATION METHODS, 46... 

ANATOMY AND CELL BIOLOGY OL DROSOPHILA OOGENESIS A BRIEL SUMMARY, 68 METHODS TO VISUALIZE OOGENESIS AND EEMALE MEIOSIS, 71 IMAGING WHOLE-MOUNT EIXED EGG CHAMBERS, 71... 

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