Wandering third-instar larvae of both sexes from various genotypes were dissected and fixed in Bouin’s fixative for 15 min and processed for immunohistochemistry as previously described (Chen et al., 2012 (link)). Confocal images of all genotypes of larvae belonging to the same experimental group were acquired using the same settings with a Zeiss LSM710 confocal microscope and image editing was done using Adobe Photoshop. Primary antibodies used were FITC-conjugated anti-HRP (1:250, Jackson ImmunoResearch Laboratories), guinea pig anti-Ringer (1:250, Mino et al., 2016 (link)), anti-Ac-Tub (1:1000, T7451, Sigma), anti-Tubulin (1:1000, 2144S, Cell Signaling), and mouse monoclonal anti-Dlg (1:1000, 4F3), anti-Brp (1:250; NC82), anti-Futsch (1:1000; 22C10), and anti-GluR IIA (1:250, 8B4D2) were obtained from Developmental Studies Hybridoma Bank (DSHB), University of Iowa. Secondary antibodies conjugated to Alexa 488 and 568 (Invitrogen-Molecular Probes) were used at 1:400 dilution.
Anti brp
Manufactured by Developmental Studies Hybridoma Bank
Sourced in United States, United Kingdom
Anti-Brp is a monoclonal antibody produced by the Developmental Studies Hybridoma Bank. It is designed to detect the Bruchpilot (Brp) protein, which is a critical component of the active zone in presynaptic terminals of neurons.
Automatically generated - may contain errors
Lab products found in correlation
Fitc conjugated anti hrp, by Jackson ImmunoResearch (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Anti ac tub, by Merck Group (1 mentions)
Anti tubulin, by Cell Signaling Technology (1 mentions)
Anti futsch, by Developmental Studies Hybridoma Bank (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Anti dlg, by Developmental Studies Hybridoma Bank (1 mentions)
Dylight conjugated goat anti hrp antibody, by Jackson ImmunoResearch (1 mentions)
Anti tubulin e7, by Developmental Studies Hybridoma Bank (1 mentions)
Anti gfp, by Abcam (1 mentions)
Anti ptauser202 thr205 at8, by Thermo Fisher Scientific (1 mentions)
Vectashield antifade mounting medium, by Vector Laboratories (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Lsm 510, by Zeiss (1 mentions)
Efficient navigation zen , by Zeiss (1 mentions)
Lsm710 confocal microscope, by Adobe (1 mentions)
Alexa 488 and 568, by Thermo Fisher Scientific (1 mentions)
5 protocols using anti brp
1
Immunohistochemistry of Drosophila Larvae
2
Drosophila Larval Neuromuscular Junction Staining
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Larvae were dissected and stained as described previously[27 (link), 67 (link)]. Following primary antibodies were used: anti-BRP (1:250)[16 (link)], anti-Tubulin (E7) (1:100) (obtained from the Developmental Studies Hybridoma Bank), anti-GFP (1:500)[41 (link)] (obtained from abcam), anti-DVGLUT (1:10,000)[19 (link)](gift from Aaron Diantonio, Washington University Medical School), anti-Liprin-α (1:500)[68 (link)](gift from Stephan Sigrist, Free University Berlin), anti-DAB [24 (link)](gift from Richard Ordway, Pennsylvania State University), and anti-dTau (1:1000)[34 (link), 35 (link)](gift from Doris Kretzschmar, Oregon Health and Science University and Daniel St. Johnston, University of Cambridge). Dylight conjugated goat anti-HRP antibody (1:1,000), Goat Cy3-, and Alexa 488 conjugated secondary antibodies against mouse, rabbit, and chicken IgG (1:1000) were obtained from Jackson ImmunoResearch, West Grove, PA.
3
Immunohistochemistry of Drosophila Brain and Salivary Gland
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Fly brains with attached lamina were dissected as previously described (Brazill et al., 2018 (link)). Salivary glands were dissected from wandering third-instar larvae (L3). Samples were fixed in freshly made 4% formaldehyde for 15 min, washed in PBS containing 0.4% (v/v) Triton X-100 (PBTX), and incubated with primary antibodies at 4°C overnight. Samples were then washed with PBTX and incubated with secondary antibodies at room temperature for 2 hr. After that, samples were stained with 4’,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific, Carlsbad, CA, USA) for 10 min and mounted with VECTASHIELD Antifade Mounting Medium (Vector Laboratories Inc, Burlingame, CA, USA). Samples were kept at 4°C until imaging. The following antibodies were used in this study: anti-Brp (1:250, Developmental Studies Hybridoma Bank, East Iowa City, IA, USA), anti-pTauSer262 (1:250, Santa Cruz Biotechnology, CA, USA), anti-pTauSer202/Thr205 (AT8, 1:250, Thermo Fisher Scientific, Carlsbad, CA, USA), and anti-Drosophila Nmnat (1:500; Zhai et al., 2006 (link)).
4
Larval Neuromuscular Junction Imaging
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Third instar larvae were dissected in ice-cold phosphate buffered saline (PBS) then fixed in 4% paraformaldehyde. After permeabilization with PBS-0.1% Triton (PBS-T) and blocking with PBS-T containing 0.2% bovine serum albumin and 2% normal goat serum, larval fillets were incubated at 4°C overnight in solutions of primary antibody. The following antibody dilutions were used: AH6 anti-PrP (TSE Reagent Resource Centre, Compton, UK) 1:1000 dilution; anti-vGlut C-terminus (kind gift from Hermann Aberle, University of Düsseldorf) 1:2000 dilution; NC82 (supernatant) anti-Brp (Bruchpilot; Developmental Studies Hybridoma Bank) 1:200 dilution and α-tubulin 1:4000 dilution. After 3 × 10 min washes in PBS-T, larvae were incubated with AlexaFluor 488 goat anti-rabbit and/or AlexaFluor 546 goat anti-mouse 1:1000 dilution for 90 min at room temperature. Larvae were mounted using Vectashield mounting medium (Vector Labs) and NMJ 6/7 (segments A2 and A3) images were acquired with a Zeiss laser-scanning confocal microscope (LSM 510, Carl Zeiss International). Image analysis was performed with ZEN (Carl Zeiss International) and Volocity software. Mean number of active zones per NMJ was calculated by dividing the number of NC82 (anti-Brp) puncta per NMJ by the total NMJ area detected by the vGlut antibody from maximum projections of z-stack images.
5
Drosophila Larval Neuromuscular Junction Immunostaining
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