All Drosophila experimental procedures were conducted with ethical approval from the Animal Care and Use Committee of Tsinghua University. Female Drosophila brains were dissected and fixed in 4% paraformaldehyde (PFA, Cat# AR-0211, Dingguo Biotech, China) at room temperature for 30 mins on a shaker. Each brain was rehydrated with 0.3% Triton X-100 (Solarbio 524A0513) in phosphate-buffered saline (PBS) for 4 × 20 mins at room temperature and then incubated in block solution (5% goat serum in washing buffer) for 30 mins at room temperature. The brain was then incubated overnight with primary antibody (mouse monoclonal nc82 (Developmental Studies Hybridoma Bank)), which was diluted at 1:500 in block solution at 4 °C. The brain was then washed in 0.5% PBST for 3 × 1 h at room temperature. Finally, the brain was mounted directly for imaging by LFM.
Mouse monoclonal nc82
Manufactured by Developmental Studies Hybridoma Bank
The Mouse monoclonal nc82 is an antibody produced by the Developmental Studies Hybridoma Bank. It is a tool used in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510 confocal laser scanning microscope, by Zeiss (2 mentions)
Cy3 conjugated goat anti rabbit, by Thermo Fisher Scientific (2 mentions)
Rabbit anti gfp, by Thermo Fisher Scientific (2 mentions)
Goat anti mouse cy5, by Jackson ImmunoResearch (2 mentions)
Goat anti chicken alexa488, by Abcam (2 mentions)
Rabbit anti rfp, by Abcam (2 mentions)
Chicken anti gfp, by Abcam (2 mentions)
Goat anti mouse alexa 488, by Thermo Fisher Scientific (1 mentions)
Lsm 710 inverted confocal laser scanning microscope, by Zeiss (1 mentions)
Mouse anti gfp, by Thermo Fisher Scientific (1 mentions)
Lsm 710 confocal laser scanning microscope, by Zeiss (1 mentions)
6 protocols using mouse monoclonal nc82
1
Drosophila Brain Dissection and Imaging
2
Immunofluorescence Staining of Whole Mount Brains
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Immunofluorescence on whole mount brains and thoracic ganglia was performed as described previously29 (link). The primary antibodies were mouse monoclonal nc82 (1:10 dilution; Developmental Studies Hybridoma Bank), rabbit anti-GFP (1:200, Invitrogen A-6455). The secondary antibodies were Alexa Fluor 488- and Cy3- conjugated goat anti-rabbit or anti- mouse IgG respectively (Molecular Probes and Jackson ImmunoResearch) diluted 1:250. Microscopy was performed using an LSM 510 laser scanning confocal microscope (Zeiss).
3
Immunofluorescence Imaging of Larvae and Adult Flies
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Immunofluorescence on peripheral and central tissues from larvae and adult flies was performed following standard procedures28 (link),45 (link). Primary antibodies: rabbit anti-IR25a (1:500)14 (link), guinea pig anti-IR25a (1:200)21 (link), mouse anti-GFP (1:500; Invitrogen), chicken anti-GFP (1:500; Abcam), rabbit anti-RFP (1:500; Abcam) and mouse monoclonal nc82 (1:10; Developmental Studies Hybridoma Bank). Secondary antibodies (all diluted 1:100–200): goat anti-mouse Alexa 488 (Invitrogen), goat anti-rabbit Cy3 (Milan Analytica, AG), goat anti-chicken Alexa488 (Abcam), goat anti-guinea pig Cy5 (Abcam) and goat anti-mouse Cy5 (Jackson ImmunoResearch). Images were collected with a Zeiss LSM 710 inverted laser scanning confocal microscope (Zeiss, Oberkochen, Germany), and processed with ImageJ and Fiji.
4
Immunofluorescence Staining of Whole Mount Brains
5
Whole-mount Immunofluorescence of Proboscis and Brain
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Immunofluorescence on whole-mount labella was performed adapting a protocol for whole-mount antennae36 (link). In brief, proboscides were dissected and fixed for 20 min in 4% PFA in PBS+0.2% Triton X-100 (PBT) at 4 °C. All washes were performed at least three times in PBT at room temperature. Primary and secondary antibody incubations were for 48 h each in PBT+5% inactivated goat serum at 4 °C. Immunofluorescence on whole-mount brains was performed following a standard protocol37 (link), except that flies were fixed for 3 h at 4 °C. Primary antibodies: rabbit anti-NOMPC (ref. 14 (link)), mouse monoclonal nc82 (diluted 1:10; Developmental Studies Hybridoma Bank), chicken anti-GFP (1:1,000; Abcam), rabbit anti-RFP (1:1,000; Abcam). Secondary antibodies: Alexa488- and Cy3-conjugated goat anti-rabbit or anti-mouse IgG, respectively (1:100; Molecular Probes and Jackson ImmunoResearch), goat anti-chicken Alexa488 (1:100; Abcam) or goat anti-mouse Cy5 (1:100; Jackson ImmunoResearch). Microscopy was performed using an LSM 710 laser scanning confocal microscope (Zeiss) and images were processed with ImageJ.
6
Drosophila Brain Immunostaining Protocol
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All Drosophila experimental procedures were conducted with ethical approval from the Animal Care and Use Committee of Tsinghua University. Female Drosophila brains were dissected and fixed in 4% paraformaldehyde (PFA, Cat# AR-0211, Dingguo Biotech, China) at room temperature for 30 mins on a shaker. Each brain was rehydrated with 0.3% Triton X-100 (Solarbio 524A0513) in phosphate-buffered saline (PBS) for 4×20 mins at room temperature and then incubated in block solution (5% goat serum in washing buffer) for 30 mins at room temperature. The brain was then incubated overnight with primary antibody (mouse monoclonal nc82 (Developmental Studies Hybridoma Bank)),
which was diluted at 1:500 in block solution at 4 °C. The brain was then washed in 0.5% PBST for 3×1 hour at room temperature. Finally, the brain was mounted directly for imaging by LFM.
which was diluted at 1:500 in block solution at 4 °C. The brain was then washed in 0.5% PBST for 3×1 hour at room temperature. Finally, the brain was mounted directly for imaging by LFM.
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