In situ hybridization
In situ hybridizations were based on a modified method of Seydoux and Fire (1994, Development 120, 2823-2834; 1995, Methods in Cell Biology) (H. Tabara and Y. Kohara, personal communication), which we modified further. Wild-type embryos were washed from 60 mm plates with M9 buffer and treated for 6-10 min with a 0.5M NaOH, 5% bleach solution to release the eggs. Intact embryos were purified by flotation on 1M sucrose (Goh and Bogaert, 1991, Development 111, 667-681). Purified embryos were resuspended in 100 l of M9 to which 100 l of 1 mg/ml chitinase (Sigma, St. Louis, MO) dissolved in 0.3M mannitol, 50mM HEPES (pH 7.2), 10mM NaCl, 10mM MgCl2, 2mM DTT was added and shaken vigorously in a microcentrifuge tube for 70 sec. The embryos were then washed three times in EH buffer (0.3M mannitol, 50mM HEPES [pH 7.2], 10mM NaCl, 10mM MgCl2, 2mM DTT, 2mM NH4NO3, 1mM EGTA, 0.1% gelatin) and once in Basal EH buffer (0.3M mannitol, 50mM HEPES [pH 7.2], 10mM NaCl, 10mM MgCl2). 50 l of Basal EH buffer was placed into a 14x14 mm poly-L-lysine-coated well (Cel-Line Associates Inc., Newfield, NJ) to which 30 l of embryos resuspended in Basal EH buffer were added and allowed to settle 8-10 min at 4°C. The excess buffer was removed and the slides were immersed in methanol (MeOH) at -20°C for 5 min. The embryos were rehydrated and fixed by immersing the slides in the follow series of solutions at 4°C: MeOH for 5 min, 7:3 (MeOH:Fixative Solution [1X PBS, 0.08M HEPES (pH 6.9), 1mM EGTA, 3.7% formaldehyde]) for 2 min, 1:1 (MeOH:Fixative Solution) for 2 min, 3:7 (MeOH:Fixative Solution) for 2 min and Fixative Solution for 20 min. The slides were then washed twice in PTw (1X PBS, 0.1% Tween 20) for 5 min, immersed in 0.2N HCl for 20 min at room temperature and washed twice again in PTw. The embryos were then immersed in Proteinase K (10 g/ml in PTw) for 11 min at room temperature, and the digestion was stopped by immersing the slides in 2 mg/ml glycine in PTw for 2 min followed by two washes in PTw. The embryos were then refixed in Fixative Solution for 20 min at room temperature, washed twice in PTw, immersed in 2 mg/ml glycine in PTw for 5 min and washed once in PTw.
Digoxygenin-labeled single stranded DNA probes for hybridization were prepared as described by Seydoux and Fire (1994, 1995). The templates for both the anti-sense lin-44 probe and the control sense lin-44 probe were deletion derivatives of the lin-44 cDNA that lacked the poly-A tail. Probes were used at a concentration of 2.5-5 g/ml in hybridization buffer. Hybridization buffer consisted of 50% formamide, 5X SSPE, 100 g/ml autoclaved salmon sperm DNA, 50 g/ml heparin, 0.1% Tween 20. Fixed embryos on slides were pre-hybridized and hybridized as described by Seydoux and Fire (1994, 1995). After hybridization, embryos were washed with slight agitation in 1:1 (Hybridization buffer [without salmon sperm DNA]: PTw), once for 2 min at 48°C and twice for 10 min at 48°C. The embryos were then washed in 0.8X PBS, 0.1% 3-([3-Cholamidopropyl]dimethylammonio)-1-propanesulfonate (CHAPS) four times for 20 min each at 48°C and once in PTw for 5 min at room temperature to remove the CHAPS. Probe detection was carried out as described by Seydoux and Fire (1994, 1995).