Goat anti hrp

For gut and pericardial cell imaging, anesthetized flies were dissected in Schneider's medium and fixed for 30 min in 4% paraformaldehyde (in PBS), rinsed in PBS and then washed three times (5 min each) in wash solution: 0.1% Triton X-100 (Sigma-Aldrich) in PBS. The tissue was blocked for 60 min in blocking solution [0.1% Triton X-100 and 2% BSA (Sigma-Aldrich) in PBS] and immunostained with primary antibodies overnight at 4°C. Samples were then washed four times for 5 min each at room temperature in wash solution, incubated with secondary antibodies at room temperature for 2 h, washed again as before and them stained with DAPI (1:1000, Sigma-Aldrich). Washed tissues were mounted in slides with Vectorshield mounting media (Vector Laboratories). The primary antibody goat anti-HRP (123-165-021, Jackson ImmunoResearch Labs) at 1:500 was used with the secondary antibody donkey anti-goat Alexa 568 (Invitrogen; 1:250).

For NMJ morphological analyses, 3^rd-instar larvae were collected and dissected as detailed above without transection of the descending motor neurons. Preparations were fixed in 3.5% paraformaldehyde HL-3, permeabilized with 0.1% Triton X-100 in 1X PBS (PBST), and blocked with 0.2% BSA in PBST (PBSTB) for 2 hrs at room temperature. Preps were washed and incubated with goat anti-HRP [Jackson Laboratories] at 1: 200 in PBSTB for 2 hrs at room temperature. Primary antibodies were removed, washed in PBSTB, and incubated with Cy3-labeled donkey anti-goat in PBSTB at 1: 400 for 1.5 hrs at room temperature. Preps were washed, mounted in VectaShield [Vector Laboratories], and imaged within three days. Images were acquired with an Olympus confocal FV1000 microscope, using a 559 nm excitation laser. Z stacks of segment A2 of muscle 6/7 were taken using 1 μm steps. The Z stacks were merged using Olympus FV1000 Fluoview Viewer, and morphology determined. Ten animals were assessed per genotype, one NMJ per animal, for a total of ten NMJs per experimental condition. Boutons were defined as varicosities at least 2 μm in diameter, and branches defined as extensions containing at least 2 boutons. Images were relabeled by an independent researcher for blinded analysis. Variance within the data set was examined using a one-way ANOVA, with comparisons made using Tukey’s post hoc test.

Third instar larvae were fixed and stained according to standard protocols. Briefly, larvae fillet preps were fixed in 4% paraformaldehyde/PBS for 20 min at room temperature, and stained with the proper primary antibodies and subsequent secondary antibodies, each for 2 h at room temperature. The following antibodies were used: chicken anti-GFP (ab13970, 1:100, Abcam), goat anti-HRP (123-605-021, 1:200, Jackson ImmunoResearch), rabbit anti-RFP (600-401-379, 1:500, Rockland Immunochemicals), rabbit anti-Ca-α1D (1:50)^{41 (link)}, rabbit anti-Ca-α1D II (1:100, this study), rabbit anti-Ca-β (1:100, this study), rabbit anti-Irk1 (1:100, this study), and fluorescence-conjugated secondary antibodies (1:1000, Jackson ImmunoResearch).

Dissected Drosophila guts were fixed at room temperature for 20 min in PBS, 4% paraformaldehyde. All subsequent incubations were done in PBS, 4% horse serum, 0.2% Triton X‐100 at 4°C following standard protocols.
The following primary antibodies were used at the following dilutions: guinea pig anti‐Ret 1/1,000 (Soba et al, 2015), mouse anti‐Pdm1 1/20 [kind gift from Steve Cohen, generated by Yeo et al (1995)], mouse anti‐Wg 1/20 (4D4 Developmental Studies Hybridoma Bank), mouse anti‐Arm 1/100 (N2 7A1 Developmental Studies Hybridoma Bank), mouse anti‐Pros 1/50 (MR1A, Developmental Studies Hybridoma Bank), chicken anti‐beta galactosidase 1/200 (ab9361, Abcam), goat anti‐HRP 1/600 (123‐095‐021 Jackson ImmunoResearch), rabbit anti‐p‐Src 1/100 (44660G; Invitrogen), rabbit anti‐pH3 (Ser10) 1/500 (9701L, Cell Signalling Technology), mouse anti‐GFP 1/1,000 (11814460001, Roche), rabbit anti‐V5 1/200 (V8137, Sigma) and rabbit anti‐phospho‐FAK (Tyr397) 1/250 (3283, Cell Signalling Technology).
Fluorescent secondary antibodies (FITC‐, Cy3‐ and Cy5‐conjugated) were obtained from Jackson Immunoresearch. Vectashield with DAPI (Vector Labs) was used to stain DNA.

The immunostaining procedure of whole-mount embryos and dissected wandering third-instar larvae were described previously (46 (link)). In brief, after fixation in 4% paraformaldehyde (for larvae) or in methanol (for embryos) and blocking by 0.5% BSA, the samples were incubated with the primary antibodies at 4 °C overnight. After washes with PBT (PBS + 0.3% Triton X-100), the samples were immunostained by the secondary antibodies at room temperature for 1 h. The following primary antibodies were used in this studies: rabbit anti-DNlg4 (1:100); mouse anti-BRP (1:50; DSHB, nc82); rabbit anti-HRP (1:1000; Jackson ImmunoResearch, West Grove, PA); mouse anti-DLG (1:50; DSHB, 4F3); rabbit anti-pMad (1:500; P. ten Dijke, Leiden University, Leiden, The Netherlands); mouse anti-CSP (1:50; DSHB, 6d6); mouse anti-SYT (1:50; DSHB, 3H2); mouse anti-GluRIIA (1:50; DSHB, 8B4D2), rabbit anti-GluRIIB (1:2000; gift from A. DiAntonio, Washington University, St. Louis, MO); rabbit anti-GFP (1:500; Santa Cruz Biotechnology); and goat anti-HRP (1:500; Jackson ImmunoResearch). The secondary antibodies, including Alexa 488- or 568-conjugated anti-mouse, anti-rabbit, or anti-goat (Invitrogen), were used at 1:500. All images were collected using a Carl Zeiss LSM 510 confocal station and analyzed with ImageJ software (National Institutes of Health).