PhD thesis - Biologisk Institut - Københavns Universitet

21 Material and methodsCCGAGTCACTGGCTCTGACAGATAGACAAGTTAAGATCTGGTTTCAGAACAGCAGCATGAATTCCGGATCCCTGGAATTGGAGAAGGAATTCGAAAGGCAACAATACATGGTTGGATCTGAAAGGTATTATCTGGCAGTATCGCTTAATTTATCCGAATCCCAAGTGAAGATCTGGTTTCAGAACCGCCGCATGAATTCCWhole mount in situ hybridisationEmbryos, larvae, and juveniles of Themiste pyroides were anesthetised by addingdrops of a 3.5 or 7% MgCl 2 solution to the seawater, and were subsequently fixed in4% paraformaldehyde (PFA) in 0.1 M phosphate-buffered saline (PBS) (pH 7.3) for20-30 min at room temperature. Subsequently, the fixative was removed by threewashes in 70% ethanol (15 min each), followed by the storage of the specimens at -20°C.Fixed specimens were rehydrated in PBS, washed four times for 5 min in PTw(phosphate-buffer containing 0.1% Tween), and permeabilised with proteinase K(Sigma; 10 µg/ml in PTw for 15 min at room temperature). Digestion was terminatedby two washes (5 min each) with PTw containing 2mg/ml of glycine and treated withtriethanolamine (TEA, 0,1M pH 7.6, Fluka; 3 x 5 min), following addition of 4µl/mland 8µl/ml of acetic anhydride without changing the TEA solution to block positivecharges. After two 5 min washes in PTw, specimens were post-fixed in 3.7% PFA for1 hr at room temperature. Subsequently, specimens were rinsed 5 x 5 min in PTw,incubated for 10 min in 50% pre-hybridisation buffer (50% formamide, 5x SSC,1mg/ml yeast RNA, 0.1 mg/ml heparin, 0.1% Tween 20, 10mM DTT) in PTw and anincubation in 100% pre-hybridisation buffer overnight at 65 °C. Antisense and sensedigoxigenin-labelled riboprobes were generated with a RNA labeling kit (SP6/T7;Roche; Copenhagen, Denmark), and used at a working concentration of 1 ng/µl forTp-mhc and Tp-actin (fragment sizes ca. 700 and 500 bp, respectively). Riboprobeswere warmed up in hybridisation buffer (50% formamide, 5x SSC, 0.1% Tween 20)for 10 min at 70 °C. Afterwards, specimens were added to that solution and incubatedfor 72 hr at 65 °C. After hybridisation, probes were recovered and specimens werewashed at 65 °C (30 min in 100% hybridisation buffer, in 75% hybridisation bufferand 75% 2x SSC, in 50% hybridisation buffer and 50% 2x SSC, in 25% hybridisationbuffer and 25% 2x SSC, 2 x 15 min in 2x SSC, and final 2 x 15 min washed in 0.2xSSC). Afterwards, specimens were washed two times 5 min in maleic buffer(MABTw; 100 mM maleic acid, 150 mM NaCl, 0.1% Tween 20, pH 7.5), blocked for