Brains of 7 day old female and male flies were dissected in PBS and directly transferred to 4% PFA with 0.1% Triton-X (PBST) for fixation over 2 hours on ice. After three 15 minute washes with PBST and blocking in 5% normal goat serum (NGS) in PBST, the primary antibodies mouse anti-brp (1:30, Developmental Studies Hybridoma Bank) and rabbit anti-GFP (1:500, Thermo Fisher) were applied in PBST with 5% NGS for 4 days nutating at 4°C. Next, the brains were washed four times for 15 minutes with PBST, blocked again with 5% NGS in PBST and incubated with the secondary antibodies goat anti-mouse Alexa Fluor 633 and goat anti-rabbit Alexa Fluor 488 (1:250 each, Thermo Fisher) in PBST with 5% NGS for 5 days nutating at 4°C. After four final washing steps with PBST, brains were mounted in VectaShield (Vector Labs). Stacks of immunostained brains were scanned on a Zeiss cLSM 880 confocal microscope with a z-step size of 0.44 μm. The confocal stacks were manually reconstructed as label fields in AMIRA (6.7, Thermo Fisher) and identified on the basis of the published Drosophila melanogaster atlases [51 (link)]. Surface renders were created and smoothed in AMIRA with the surface gen and smooth surface tools.
Mouse anti brp
Manufactured by Developmental Studies Hybridoma Bank
Sourced in United Kingdom, United States
Mouse anti-brp is a monoclonal antibody that specifically binds to the Bruchpilot (Brp) protein in Drosophila. Brp is a key component of the active zone, a specialized region of the presynaptic nerve terminal where neurotransmitter release occurs. The mouse anti-brp antibody is a useful tool for immunohistochemical detection and localization of Brp in Drosophila tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti gfp, by Thermo Fisher Scientific (4 mentions)
Alexa fluor 633 goat anti mouse, by Thermo Fisher Scientific (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (2 mentions)
Clsm 880 confocal microscope, by Zeiss (2 mentions)
Tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
Cy3 conjugated anti hrp, by Jackson ImmunoResearch (1 mentions)
Ab8227, by Abcam (1 mentions)
Rabbit anti ha, by Cell Signaling Technology (1 mentions)
Rat anti flag, by Novus Biologicals (1 mentions)
Mouse anti neuroglian, by Developmental Studies Hybridoma Bank (1 mentions)
Mouse anti repo, by Developmental Studies Hybridoma Bank (1 mentions)
Chicken anti gfp, by Abcam (1 mentions)
A11008, by Thermo Fisher Scientific (1 mentions)
A 11030, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse alexa 546, by Thermo Fisher Scientific (1 mentions)
Sp5 2 confocal microscope, by Leica (1 mentions)
Rabbit anti dsred, by Takara Bio (1 mentions)
Lsm 510 meta microscope, by Zeiss (1 mentions)
7 protocols using mouse anti brp
1
Drosophila Brain Immunostaining and Confocal Imaging
2
Western Blot Analysis of Fly Proteins
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The antibodies used here were rabbit anti-β actin (ab8227; Abcam), mouse anti-GFP (11 814 460 001; Roche), rabbit anti-Rab7 (Tanaka and Nakamura, 2008 (link)), rabbit anti-Arl8 (Hofmann and Munro, 2006 (link)), Cy3-conjugated anti-HRP (123-165-021; Jackson ImmunoResearch, Ely, UK), rabbit anti-Syt (West et al., 2015 (link)), mouse anti-BRP [nc82; Developmental Studies Hybridoma Bank (DSHB)], mouse anti-Notch extracellular domain (C458.2H; DSHB). Information on validation of the antibodies is available from the commercial source or the indicated publication. Primary antibodies were detected by secondary antibodies conjugated with Alexa fluorochromes (Thermo Fisher Scientific) or with HRP (DAKO).
Whole flies, larvae or the indicated dissected fly tissues were lysed in equivalent amounts of SDS sample buffer and insoluble debris was removed by brief centrifugation. S2 cells growing in plates were dissolved directly in SDS sample buffer. Protein extracts were separated in 4–20% Tris-Glycine gels (Invitrogen), transferred to PVDF membranes and probed with primary and HRP-conjugated secondary antibodies, detected by chemiluminescence (ECL; GE Healthcare).
Whole flies, larvae or the indicated dissected fly tissues were lysed in equivalent amounts of SDS sample buffer and insoluble debris was removed by brief centrifugation. S2 cells growing in plates were dissolved directly in SDS sample buffer. Protein extracts were separated in 4–20% Tris-Glycine gels (Invitrogen), transferred to PVDF membranes and probed with primary and HRP-conjugated secondary antibodies, detected by chemiluminescence (ECL; GE Healthcare).
3
Immunolabeling of Drosophila Neuromuscular Junctions
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Larvae were dissected and labeled as described previously[26 (link)] using rabbit anti-DGluRIII[26 (link)] (1:1000) and mouse anti-BRP[25 (link)] (1:250) primary antibodies (both from Developmental Studies Hybridoma Bank, Iowa City, IA). We also used mouse anti-Dlg (1:2000) (mAb 4f3), developed by Corey S. Goodman (Renovis, San Francisco, CA, USA) and obtained from the Hybridoma Bank; guinea pig anti-WASP[48 (link)] (1:1000), a gift from Dr. Scherzer (Rehovolt, Israel). Goat Cy5/dylight anti-HRP antibody (1:1000), Cy3- and mouse or rabbit Alexa 488- or Cy3- conjugated secondary antibodies and Alexa 633 or Cy5-conjugated anti-guinea pig antibody (1:1000) were obtained from Jackson ImmunoResearch (West Grove, PA, USA).
4
Immunofluorescence Staining Protocol
5
Immunohistochemistry of Drosophila Brain
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Brain immunohistochemistry was performed as previously described (Chu et al. 2014 (link)). To stain flies with the UAS-CsChrimson.mVenus transgene, the following primary antibodies were used: mouse anti-brp (1:50, Developmental Studies Hybridoma Bank #nc82) and rabbit anti-GFP (1:1000, Invitrogen), with secondary antibodies: goat anti-rabbit Alexa-488 (A11008, Invitrogen) and goat anti-mouse Alexa-546 (A11030, Invitrogen). For GRASP experiments, the following were used as primary antibodies: mouse anti-GFP (1:100, Sigma-Aldrich, G6539), rat anti-DN- cadherin (1:25, DSHB DNEX#8), and rabbit anti-DsRed (1:1000, Clontech #632496) with secondary antibodies: goat anti-mouse Alexa-488 (A11029, Invitrogen), goat anti-rat Alexa-568 (A11077, Invitrogen) and goat anti-rabbit Alexa-647 (A21245, Invitrogen).
All images were acquired using a Leica SP5 II Confocal microscope. Images taken at a magnification of 25x were with a water immersion objective with a Z-stack step size of 1 μm, while those imaged at 63x were with oil immersion and a step size of 0.5 μm.
All images were acquired using a Leica SP5 II Confocal microscope. Images taken at a magnification of 25x were with a water immersion objective with a Z-stack step size of 1 μm, while those imaged at 63x were with oil immersion and a step size of 0.5 μm.
6
Dissection and Immunolabeling of Larval Nervous System
7
Drosophila Neuroanatomy Mapping
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Brains of 7 day old female and male flies were dissected in PBS and directly transferred to 4% PFA with 0.1% Triton-X (PBST) for fixation over 2 hours on ice. After three 15 minute washes with PBST and blocking in 5% normal goat serum (NGS) in PBST the primary antibodies mouse anti-brp (1:30, Developmental Studies Hybridoma Bank) and rabbit anti-GFP (1:500, Thermo Fisher) were applied in PBST with 5% NGS for 4 days nutating at 4°C. Next, the brains were washed four times for 15 minutes with PBST, blocked again with 5% NGS in PBST and incubated with the secondary antibodies goat anti-mouse Alexa Fluor 633 and goat anti-rabbit Alexa Fluor 488 (1:250 each, Thermo Fisher) in PBST with 5% NGS for 5 days nutating at 4°C. After four final washing steps with PBST, brains were mounted in VectaShield (Vector Labs). Stacks of immunostained brains were scanned on a Zeiss cLSM 880 confocal microscope with a z-step size of 0.44 µm. The confocal stacks were manually reconstructed as label fields in AMIRA (6.7, Thermo Fisher) and identified on the basis of the published Drosophila melanogaster atlases [51] .
Surface renders were created and smoothed in AMIRA with the surface gen and smooth surface tools.
Surface renders were created and smoothed in AMIRA with the surface gen and smooth surface tools.
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