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Fig. 2. (I) Isolation of cortices from oocytes, zygotes and embryos. (IA). Eggs or synchronous embryos deposited into a drop of cortex buffer and allowed to attach to a polylysine-coated coverslip are sheared with a stream of cortex buffer from a Pasteur pipette. (IB) Fields of isolated cortices are labelled, mounted and observed using epifluorescence or confocal microscopy. (IC) The position of grey coverslips (c) indicate the origin of the cortical fragments isolated from oocytes, zygotes or embryos selected for observation. cER is red, the myoplasm is green, microtubules in light blue and DNA in dark blue. a, animal; v, vegetal; cp, contraction pole; d, dorsal; P, posterior; A, anterior. (ID) Field of cortices isolated from unfertilized eggs and labelled for cER. (II) Isolated cortices from oocytes retain a-v polarity. (IIA1) Cortex isolated from an oocyte labelled for cER (red) and microfilaments (yellow). Regions corresponding to the animal (a) and vegetal (v) hemispheres are shown. (IIA2) Detail of the region boxed in IIA1. (IIB1-B3) Cortex isolated from an oocyte. cER is double labelled with DiIC16(3) (B1, red) and by immunofluorescence for ribosomes (B2, green). Merged image of cER network and ribosomes (B3, colocalization in yellow). Dotted line indicates the edge of isolated cortex. Arrowhead indicates cytoplasmic ER that has fallen onto the coverslip during the shearing process (B3, arrowhead). (IIC1-D2) Fragments of cortices isolated from an oocyte labelled for cER (C1, red) and Ci-PEM1 (C2, green) (IID1,D2). Cortex isolated and labelled after KCl-puromycin treatment; labelled for cER (D1) and ribosomes (D2). (IIE). Replica of a fragment of an isolated oocyte cortex (fast-freezing deep-etching electron microscopy). Cortical ER is red, microfilaments orange and particles on the cER surface yellow. Most particles are the size of ribosomes (arrowheads), coated pits are blue and the internal side of the plasma membrane (PM) is grey. All cortices were obtained from Ciona oocytes except for E, which is from Phallusia.