Embryo fixation was performed according to standard methods. Embryos were fixed by a solution containing heptane (Sigma, St Louis, MO) and methanol57 (link),58 (link). Wing discs were fixed in PLP fixative (2% paraformaldehyde, 75 mM lysine, and 35 mM phosphate buffer, pH7.4) for 15–30 min at room temperature.
Antibodies used for immunohistochemistry were as follows: mouse anti-Cut (1:200, 2B10, Developmental Studies Hybridoma Bank (DSHB), Iowa City, Iowa), guinea pig anti-Centrosomin (1:1000, from Jordan Raff), Rat anti-α-Tubulin (1:200, MAB1864, Millipore, Burlington, Massachusetts), rabbit anti-PH3 (1:200, 06–570, Millipore), Rabbit anti-Vtd (1:500, this study), rabbit anti-Myc (1:100, ab9106, Abcam, Cambridge, UK). Secondary antibodies conjugated with Rhodamine Red™-X (RRX), Alexa Fluor® 647 or fluorescein isothiocyanate (1:200, 715-095-151, 715-295-151, 715-605-151, 711-095-152, 711-295-152, 711-605-152, 706-095-148, 706-295-148, 706-605-148, 712-095-153, 712-295-153, 712-605-153) were from Jackson ImmunoResearch Inc. Vectashield with 4′, 6-diamidino-2-phenylindole (H-1200, Vector Laboratories, Burlingame, CA) was used for mounting. Fluorescent images were acquired using Carl Zeiss LSM710 confocal microscope (Carl Zeiss, Oberkochen, Germany).
Antibodies used for immunohistochemistry were as follows: mouse anti-Cut (1:200, 2B10, Developmental Studies Hybridoma Bank (DSHB), Iowa City, Iowa), guinea pig anti-Centrosomin (1:1000, from Jordan Raff), Rat anti-α-Tubulin (1:200, MAB1864, Millipore, Burlington, Massachusetts), rabbit anti-PH3 (1:200, 06–570, Millipore), Rabbit anti-Vtd (1:500, this study), rabbit anti-Myc (1:100, ab9106, Abcam, Cambridge, UK). Secondary antibodies conjugated with Rhodamine Red™-X (RRX), Alexa Fluor® 647 or fluorescein isothiocyanate (1:200, 715-095-151, 715-295-151, 715-605-151, 711-095-152, 711-295-152, 711-605-152, 706-095-148, 706-295-148, 706-605-148, 712-095-153, 712-295-153, 712-605-153) were from Jackson ImmunoResearch Inc. Vectashield with 4′, 6-diamidino-2-phenylindole (H-1200, Vector Laboratories, Burlingame, CA) was used for mounting. Fluorescent images were acquired using Carl Zeiss LSM710 confocal microscope (Carl Zeiss, Oberkochen, Germany).
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