Despite the difficulty in identifying discreet bundles of photoreceptor axons, distal innervation was evident. the protein trap construct also discloses expression deeper in the brain, round the IPC and forming a mesh\like structure inside the hemisphere, where the antibody did not BMS-1166 penetrate. Inset shows expression between the LPC and lopn. Anti\E\cad staining (magenta) was used to identify the neuroepithelial cells and anti\Chaoptin (gray) label photoreceptors. (I) The driver mediated membrane labeling (green) pattern is very similar to the one observed with the antibody and the protein trap construct, including the transmission detected between the LPC and lopn. Glial nuclei werel abeled with anti\Repo antibody (blue). Not all glial nuclei are ClC\a+(reddish). (J\K) protein trap expression surrounding type I (J\J) and type II (K\K) neuroblasts Rabbit Polyclonal to ACTL6A labeled with anti\Dpn antibody (blue). (J\J) Confocal sections at different levels of a type I neuroblast (arrow) show the presence of ClC\a\GFP protein surroundingit. (K\K) Confocal sections at different levels of a type II neuroblast (arrow) show the presence of INPs (asterisks) also labeled with anti\Dpn.ClC\a\GFP is seen surrounding the neuroblast and delineating the chamber encasing the INPs and the neuroblast lineage. CB, central brain; OL, optic lobe; LF, lamina furrow; LPC, lamina precursor cells; lopn, lobula plate neurons; OPC, outer proliferation center; IPC, inner proliferation center. Level bars symbolize 10 m. GLIA-67-2374-s003.tif (12M) GUID:?4C8A51EB-34A2-4510-A5D2-126525CF8C36 Supplementary Figure 2. Identification of expressing glia. Confocal sections showing expression pattern in the late L3 nervous system (A\D), and the optic lobe in pupal stages (E, F) and adult (G). specific GAL4 driver was used to label cellular membranes (green) and nuclei (red) of ClC\a+ cells. Glial nuclei were labeled with anti\Repo antibody (blue) and photoreceptor cells with BMS-1166 anti\Chaoptin (gray). (A) Larval brain where, besides a ClC\a+ transmission in cortex glia both in brain hemispheres and the VNC, a ClC\a+ transmission is detected in neuropil\ensheathing glia in the VNC, tract\ensheathing glia in connectives between the two hemispheres, and in peripheral nerves. (B,C) Cross section (B) and longitudinal section (C) of peripheral nerves made up of ClC\a+ glia. Dashed collection outlines BMS-1166 the nerve. (D) Image of the optic stalk, which connects the eye disc and the optic lobe. ClC\a+ glia wraps this bundle created by photoreceptor axons on their way to the optic lobe. Photoreceptor cell body are seen in the eye disc in gray and their axons in the optic lobe. Photoreceptors do not express expressing glia: cxg, wg/dsg, Xgo, Xgi,mneg, and lopneg in 20 (E) and 50 (F) hrs After Pupal Formation (APF). (G) expression is managed in the adult. Transmission in the medulla and lobula neuropils belongs to mneg and lopneg explained projections into these structures. egt, tract\ensheathing glia; egn, neuropil\ensheathing glia; wg, wrapping glia; pn, peripheral nerve; ed, vision disc; os, optic stalk; OPC, outer proliferation center; LPC, lamina precursor cells; BBB, blood brain barrier; cxg, cortex glia; megn, medulla neuropil\ensheathing glia; ep, epithelial glia; mg, marginal glia; Xgo, outer chiasm glia; Xgi, inner chiasm glia; psg, proximal BMS-1166 satellite glia; wg/dsg, wrapping glia/distal satellite glia; lopegn, lobula plate neuropil\ensheathing glia. Level bars symbolize 10 m. GLIA-67-2374-s004.tif (5.7M) GUID:?6A6EF540-6443-4ADE-80A9-43FEA6895F6D Supplementary Physique 3. Immunohistochemistry and western blot analysis of MiMIC alleles. BMS-1166 (A, B) Anti\ClC\a antibody staining of adult Malpighian tubules in control animals (A) and mutants (B). (C, D) Anti\ClC\a antibody staining of late L3 brains in control animals (C) and mutants (D). Photoreceptors are labeled with anti\Chaoptin (green).(E) Western blot of protein extraction from HEK293 cells transfected with or without pcDNA3.1. Both anti\Flag and anti\ClC\a antibodies detect a band below 130 kDa, which is usually possibly the excess weight of the protein (Uniprot prediction at 118 kDa) plus glycosylation. (F) Western blot of protein extraction from adult heads of controls and different allelic combinations. The transmission round the 130 kDa mark reflects.