Mouse anti prospero mr1a

Immunohistochemistry of Drosophila tissue was performed using standard procedures. Briefly, larva or adult intestine were dissected in ice-cold PBS and fixed in either 80% ice-cold methanol in PBS for 1 h on ice (for staining with anti-LbCas12a antibody) or in 4% paraformaldehyde in PBS containing 0.05% Triton-X 100 for 25 min at room temperature (for staining with any of the other antibodies). Larva were washed three times in PBS containing 0.3% Triton-X 100 (PBT) and then blocked for 1 h at room temperature in PBT containing 1% heat-inactivated normal goat serum. Subsequently, larva were incubated with first antibody [mouse anti-LbCas12a (Sigma Aldrich) 1:20; mouse anti-Cut (Gary Rubin, Developmental Studies Hybridoma Bank) 1:30; rabbit anti-Evi (42 (link)) 1:800; mouse anti-Prospero (MR1A, C. Q. Doe, Developmental Studies Hybridoma Bank) 1:1,000] in PBT overnight at 4 °C. The next day, samples were washed three times in PBT for 15 min and incubated for 2 h at room temperature with secondary antibody (antibodies coupled to Alexa fluorophores, Invitrogen) diluted 1:600 in PBT containing Hoechst dye. Samples were washed three times 15 min in PBT and mounted in Vectashield (Vectorlabs). To visualize apoptotic cells, wing discs expressing the apoptosis sensor GC3Ai (43 (link)) were fixed in 4% PFA, washed in PBT containing Hoechst and mounted in Vectashield.

Late third instar larval brains were dissected in PBS and fixed with 4% PFA/PBS/0.3%Triton for 20 min. For immunostaining, brains were blocked in PBS/0.3%Triton/1%BSA/5% normal goat serum and incubated in primary antibody in PBS/0.3%Triton/1%BSA overnight. Primary antibodies include rat anti‐Deadpan (Abcam Cat# ab195172, 1:250 or 1:500, Waltham, MA, USA) and mouse anti‐Prospero MR1A (Developmental Studies Hybridoma Bank, 1:1000, Iowa City, IA, USA). The donkey secondary antibodies were used at 1:500 (Jackson ImmunoResearch, West Grove, PA). Brains were mounted with double sided tape spacers and imaged on a Leica Sp8 confocal with 2.0 μm sections throughout the entire brain lobe using a 25× water immersion lens. Brain lobe volumes were quantified using Imaris (Bitplane) and the Volume function (units are μm³). Volume was then normalized to Ankle2^A; hANKLE2 wt rescue animals. Rescue animals were set to 100%. Mutant and variant rescue animals are calculated as percent (%) of rescue volume for display purposes. Number of animals tested are as follows: hANKLE2 wt (6), Ankle2^A (8), Vall229Gly (10), Arg236* (5), Arg536Cys (8), and Gly510* (8). hANKLE2 p.Arg236* animals (n = 5) were difficult to obtain due to lower‐than‐expected Mendelian ratios. Note that Ankle2^A is a temperature sensitive mutation, and slight fluctuation in temperature can affect severity of brain volume defects.