Goat anti mouse fitc

For immunolabeling of motor axons, embryos were first subject to an antigen retrieval step by incubation in 150 mM Tris HCl pH 9 for 15 minutes at 70 °C, washed two times in 0.1% PBS-Tween 20 in then incubated in acetone at −20 °C for 15 minutes. Embryos were washed three times in 0.1% PBS-Tween 20 incubated then blocked for 1 hour at room temperature (RT) with 10% normal serum, 0.8% Triton X-100, 1% BSA in 0.1% PBS-Tween 20, followed by overnight incubation at 4 °C with primary antibody znp-1 (1:200, Developmental Studies Hybridoma Bank Cat# znp-1, RRID:AB_2315626) to label motor axons and branching. The primary antibody was removed from embryos by washing two times with 0.8% PBS-Triton X-100 and three times with PBS-TS (1% Triton, 10% normal serum, 1 × PBS) and embryos were then incubated in secondary antibody FITC goat anti-mouse (1:500, Jackson Laboratories cat # 115-096-006) for 2 h at RT. Embryos were washed twice in PBS-TS and stored in 1X PBS at 4 °C until flat mounting.

Immunofluorescence was mainly performed as described^{56 (link)}. Briefly, freshly dissected bones were fixed in 4% paraformaldehyde for 48 h and incubated in 15% DEPC-EDTA (pH 7.8) for decalcification. Then, specimens were embedded in paraffin or OCT and sectioned at 8 μm. Sections were blocked in PBS with 10% horse serum for 1 h and then stained overnight with mouse-anti-Osteocalcin (Santa Cruz, 1:100, sc-376726), rabbit-anti-Ddah1 (SAB, 1:200, #37368), mouse-anti-Ddah1 (Santa Cruz, 1:100, sc-271337), rabbit-anti-Ddah2 (SAB, 1:200, #38934), mouse-anti-TAZ (Abcam, 1:200, ab242313), rabbit-anti-YAP (Abcam, 1:200, ab52771), and eNOS (Santa Curz, 1:200, sc-376751). Goat-anti-mouse FITC (1:1000; Jackson ImmunoResearch, 705-165-147) and donkey-anti-rabbit Alexa Fluor 488 (1:1000; Molecular Probes, A21206) were used as secondary antibodies. DAPI (Cell Signaling Technology, #4083) and DyLight™ 594 Phalloidin (Cell Signaling Technology, #12877) were used for counterstaining. All immunofluorescence experiments were confirmed by at least one independent repeat. An Olympus IX81 confocal microscope or Zeiss LSM-880 confocal microscope was used to image samples.

Either last-instar larval or 12–24 h pupal wing discs were fixed for 30 min in 0.1 M PIPES (pH 6.9), 1 mM EGTA, 1% Triton X-100, 2 mM MgSO₄, and 1.8% formaldehyde. The discs were then incubated in 50 mM Tris (pH 6.8), 150 mM NaCl, 0.5% NP40, and 5 mg/ml bovine serum albumin (BSA) (block buffer) for a minimum of 2 h at 4 °C. The wings were then placed in 50 mM Tris (pH 6.8), 150 mM NaCl, 0.5% NP40, and 1 mg/ml BSA (wash buffer) containing either rabbit anti-Spalt (1:200), or mouse anti-En/Inv (4F11) (1:5)/rat anti-Ci (1:25)/rabbit anti-Dll (1:100) and incubated overnight at 4 °C. The wings were washed 4 times in wash buffer and then incubated for 2 h at 4 °C in wash buffer containing goat anti-mouse FITC (1:200, Jackson Laboratories, West Grove, PA), goat anti-rat Cy3 (1:200, Jackson Laboratories, West Grove, PA), and goat anti-rabbit Cy5 (1:200, Jackson Laboratories, West Grove, PA). The wing discs were washed four times in wash buffer and then placed on glass slides with the Vectashield (Vector Laboratories, Burlingame, CA). Glass coverslips were applied over the discs and images were collected on a MRC600 laser-scanning confocal microscope. Images were individually collected and then assembled using Adobe Photoshop (Adobe Systems Incorporated, San Jose, CA) software.

Midguts were dissected from flies at 10, 20 and 40 days, fixed in 4% paraformaldehyde and stained as described [17 (link)]. Following washing, samples were mounted and imaged using the Leica camera attachment using LAS V4.1 software, or the Zeiss 780 combined confocal/FCS/NLO system, mounted on an inverted Axio Observer Z1. Fixed tissue was incubated overnight with mouse anti-gal 1:500 (Invitrogen); rabbit anti-pH3 1:300 (Invitrogen) or rabbit anti-INDY 1:300 [10 (link)] primary antibodies diluted in PBT [0.1%Triton X-100 in phosphate-buffered saline (PBS)] at 4°C. Following washing and blocking, tissue was incubated with the goat anti-rabbit Cy3 1:300 (Jackson) or goat anti-mouse FITC 1:300 (Jackson) secondary antibodies and DAPI 1:1000 (Invitrogen) diluted in PBT and 2% donkey serum for 1 hour at room temperature. Images were analyzed using Adobe Photoshop or Image J. Variability between different regions of the gut was reduced by quantifying images from the same designated region for each genotype in a 0.06x 0.02cm area. Cells were counted, values averaged and standard deviation calculated separately.

Primary antibodies used were rabbit anti-K63 ubiquitin (Apu3 from Millipore), rabbit anti-K48 ubiquitin (Apu2 from Millipore) and mouse monoclonal anti-1CB4 (generous gift from Steve L’Hernault at Emory University). Secondary antibodies used in immunofluorescence were goat-anti-mouse FITC and goat-anti-rabbit TRITC (Jackson ImmunoResearch Laboratories).

The following primary antibodies diluted in PBST were used in these experiments: mouse anti-Delta, mouse anti-Arm (Developmental Studies Hybridoma Bank, Iowa City, IA, USA), 1:200; mouse anti-GFP and rabbit anti-GFP (Molecular Probes, Eugene, OR, USA), 1:1000; rat anti-GFP (Nacalai Tesque Inc., Kyoto. Japan), 1:1000; rabbit anti-γH2AvD (Rockland, Gilbertsville, PA, USA) 1:2000; rabbit anti-pS/TQ (Cell Signaling Technologies, Danvers, MA, USA), 1:1000; rabbit anti-phospho-histone H3 (PH3, Millipore, Billerica, MA, USA), 1:1000; mouse anti-γ-tubulin (Sigma-Aldrich), 1:1000; rabbit anti-β-gal (Upstate Biotechnology Inc., Lake Placid, NY, USA), 1:1000; and anti-CCleaved caspase-3 (Cell Signaling Technologies), 1:1000; rabbit anti-pJNK antibody (Cell Signaling Technologies). The following secondary antibodies diluted in PBST were used: goat anti-rabbit FITC (Jackson ImmunoResearch, West Grove, PA, USA), 1:400; goat anti-rabbit Cy3 (Jackson ImmunoResearch), 1:400; goat anti-mouse FITC (Jackson ImmunoResearch), 1:400; goat anti-mouse Cy3 (Jackson ImmunoResearch), 1:400; goat anti-rat FITC (Jackson ImmunoResearch), 1:400, goat anti-rabbit Alexa Fluor^® 647 (Jackson ImmunoResearch), 4′,6-diamidino-2-phenylindole (DAPI, Molecular Probes), 1:1000.