Flies were anesthetized on ice and placed inside cut pipette tips allowing the head to protrude at the narrow end. One of the maxillary palps or antennae was excised and neurobiotin crystals (Vector Laboratories, United States) were immediately applied to the fresh wound. The preparation was left in a humidified chamber overnight at 7°C. The following day, the brain was dissected out in 0.01 M phosphate-buffered saline (PBS) and fixed overnight in 4% paraformaldehyde diluted in PBS at 7°C. After careful rinsing in PBS, the brains were incubated for 72 h in mouse monoclonal anti-synapsin (anti-SYNORF1, 1:20; Developmental Studies Hybridoma Bank, Iowa City, IA, United States) and Alexa Fluor 488 conjugated streptavidin (1:500, Life Technologies, Netherlands). The brains were then rinsed again and incubated for 48 h with Alexa 546-tagged secondary antibody (1:500; Invitrogen), to detect anti-synapsin staining. The brains were subsequently rinsed and dehydrated in increasing concentrations of ethanol and cleared in methyl salicylate. Finally, the brains were mounted in methyl salicylate with spacers under cover glasses and scanned with 10× or 20× air objectives using a Zeiss LSM 780 META confocal laser scanning microscope (Zeiss, Jena, Germany).
Anti synorf1
Manufactured by Developmental Studies Hybridoma Bank
Sourced in United States
Anti-SYNORF1 is a monoclonal antibody that recognizes the SYNORF1 protein. The SYNORF1 protein is a synaptic vesicle protein involved in neurotransmitter release. Anti-SYNORF1 can be used to detect and localize the SYNORF1 protein in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Alexa 546 tagged secondary antibody, by Thermo Fisher Scientific (2 mentions)
Anti phospho histone h3 ser10 d2c8 ph3, by Cell Signaling Technology (2 mentions)
Alexa 488 conjugated goat anti mouse, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
Neurobiotin crystals, by Vector Laboratories (1 mentions)
Goat anti mouse igg, by Merck Group (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Abcam (1 mentions)
Anti h3p, by Merck Group (1 mentions)
Tcs spe, by Leica (1 mentions)
Lsm 510 meta, by Zeiss (1 mentions)
Axiovert 200m, by Leica (1 mentions)
Eclipse 80i, by Nikon (1 mentions)
Anti α tubulin, by Developmental Studies Hybridoma Bank (1 mentions)
Anti arrestin, by Cell Signaling Technology (1 mentions)
Alexa fluor dye, by Thermo Fisher Scientific (1 mentions)
Anti hrp cy3, by Jackson ImmunoResearch (1 mentions)
Alexa 568 conjugated goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa 546, by Thermo Fisher Scientific (1 mentions)
11 protocols using anti synorf1
1
Neuroanatomy Mapping of Insect Brains
2
Immunostaining Quantification Protocol
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Immunostaining was carried out as described previously [43] (link). The following antibodies were used: anti-synapsin (anti-SYNORF1, 1∶50; Developmental Studies Hybridoma Bank), anti-Smed-β-catenin2 (1∶1000; Chai et al., 2010) and anti-phospho-histone H3 (Ser10) (D2C8) (pH3) (1∶500; Cell Signaling Technology). Images were scanned, processed and quantified as described for FISH images. To avoid technical variance and obtain a reliable quantification of pH3+ cells, at least two independent experiments of RNAi and pH3 immunostaining were carried out for anterior amputation, incision, degrowth and growth experimental designs.
3
Planarian Whole-Mount Immunostaining
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The planarians were sacrificed in ice-cold 2% HCl for 2–5 min and fixed in 4% paraformaldehyde, then rinsed in 4°C methanol for 1 h and bleached in methanol containing 6% H2O2 overnight. After rehydration, specimens were blocked in 0.6% BSA and 0.45% fish gelatin diluted with PBSTx for 1 h at room temperature and incubated sequentially in primary antibody concentrations were used as follows: anti-phospho-Histone H3 (anti-H3P, 1:250, R&D Systems, United States); anti-Acetylated Tubulin (anti-Ac-Tubulin, 1:500, Sigma, United States); anti-SYNORF1 (1:50, Developmental Studies Hybridoma Bank, United States). Secondary antibody concentrations were: goat-anti-mouse IgG (1:200, Millipore, United States), and goat anti-rabbit IgG (Alexa Fluor488) (1:1,000, Abcam, United Kingdom). The signals were photographed and fluorescent images were captured and analyzed with LEICA SP8 confocal microscope and ImageJ software.
4
Immunohistochemistry of Flatworm Markers
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Immunohistochemistry was performed after either paraformaldehyde of Carnoy fixative based fixation64 (link). Antibodies used were: anti-H3P (phosphorylated serine 10 on histone H3; Millipore; 09–797; 1:1000 dilution), anti-VC1 (kindly provided by Prof. Hidefumi Orii (check title); 1:10,000 dilution), anti-SMEDWI-1 (kindly provided by Prof. Jochen Rink; 1:500 dilution), anti-SYNORF-1 (3C11; Developmental Studies Hybridoma Bank; 1:50 dilution), anti-acetylated tubulin (Developmental Studies Hybridoma Bank; 1:200 dilution).
5
Immunohistochemistry of Regenerating Planarians
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Regenerating planarians were fixed and processed as previously described [41 (link)]. Primary antibodies used were 3C11 (anti-SYNORF-1, Developmental Studies Hybridoma Bank, dilution 1 : 50), anti-phosphoserine 10 Histone H3 (H3P, Millipore, dilution 1 : 600), and anti-SMEDWI-1 (1 : 1500; Guo et al., 2006 [42 (link)]; März et al., 2013 [43 (link)]). Secondary antibodies used were Alexa 488-conjugated goat anti-mouse (Molecular Probes, dilution 1 : 400) and Alexa 568-conjugated goat anti-rabbit (Millipore, dilution 1 : 500). To determine the mitotic activity of the stem cells, the total number of mitotic neoblasts was normalized to the body size of the animals, which was determined using ImageJ (v1.49p, National Institute of Health) by measuring the surface of the animals before sampling. Nuclear counterstaining was performed using DAPI (1 μg/mL) overnight at 4°C. The animals were examined with fluorescence microscopy performed with a Nikon Eclipse 80i (Nikon Instruments, Melville, USA) and confocal imaging was performed using a Zeiss LSM510 META (Carl Zeiss, Jena, Germany) mounted on an Axiovert 200 M (Laser: 488 nm; Filter: BP 500–550 IR; Beam splitters: MBS: HFT 488, DBS1: mirror, DBS2: NFT 490), and a Leica TCS-SPE (Leica, Heidelberg, Germany).
6
Immunostaining and Confocal Microscopy
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Immunostaining was carried out as described previously [18 (link)]. The following antibodies were used: anti-synapsin (anti-SYNORF1, 1 : 50; Developmental Studies Hybridoma Bank), anti-arrestin (VC1, a kind gift of Professor H. Orri), anti-phosphohistone H3 (Ser10) (D2C8) (pH3) (1 : 500; Cell Signaling Technology), and anti-α-tubulin (1 : 20, Developmental Studies Hybridoma Bank). Confocal laser scanning microscopy was performed using a Leica TCS 4D (Leica Lasertechnik, Heidelberg) adapted for an inverted microscope (Leitz DMIRB). Images were processed using ImageJ software. p-H3 positive cells were counted by visual inspection of confocal z-stacks of images corresponding to the whole animal. In the corresponding graphs error bars represent standard error of the mean.
7
Mosquito Head Immunostaining Protocol
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Following removal of the proboscis, mosquito heads were fixed in 4% paraformaldehyde for 1 h at room temperature23 (link). After fixation, heads were embedded in albumin/gelatin, post-fixed in 6% formaldehyde overnight at 4 °C and sectioned (40 µm). Sections were washed in phosphate-buffered saline 0.3% Triton X-100 and blocked in 5% normal goat serum and 2% bovine serum albumin. Primary antibodies used were monoclonal antibody 3C11 (anti-SYNORF1; 1:50; Developmental Studies Hybridoma Bank, University of Iowa, http://dshb.biology.uiowa.edu/ ) and the conjugated primary antibody anti-HRP-Cy3 (1:500, Jackson ImmunoResearch, Code: 123-165-021). Secondary antibodies used correspond to Alexa Fluor Dyes (1:500; Thermo Fisher). Samples were mounted in DABCO and visualised using a Zeiss 880 confocal microscope.
8
Whole-mount Immunohistochemistry of Planarian Tissues
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Whole-mount immunohistochemistry was performed as previously described [102 (link),122 (link)]. The following antibodies were used: mouse anti-SYNAPSIN, used as pan-neural marker (anti-SYNORF1, Developmental Studies Hybridoma Bank, Iowa City, IA, USA) diluted 1 : 50; mouse anti-VC1 [70 (link)], specific for planarian photosensitive cells (anti-arrestin, kindly provided by H. Orii and Professor K. Watanabe) diluted 1 : 15 000; rabbit anti-phospho-histone H3 (Ser10) to detect cells at the G2/M phase of cell cycle (H3P, Cell Signaling Technology) diluted 1 : 300; anti-RAPUNZEL-1 [78 (link)], used as a marker for intestinal goblet cells (RPZ-1, kindly provided by K. Bartscherer) used at 1 : 200. The secondary antibodies Alexa 488-conjugated goat anti-mouse and Alexa-568-conjugated goat anti-rabbit (Molecular Probes, Waltham, MA, USA) were diluted 1 : 400 and 1 : 1000, respectively. Samples were mounted in 70% glycerol before imaging.
9
Immunohistochemistry of Butterfly Brains
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Heads or the anterior body of the different stages of butterflies were fixed overnight at 4°C in 4% paraformaldehyde in 0.1 M sodium phosphate buffer (PB). After careful rinsing in PB, the brains were dissected in phosphate-buffered saline with 0.25% Triton-X (PBS-Tx). Thereafter, the brains were preincubated overnight in 5% normal goat serum in PBS-Tx. Then brain tissue was incubated for 72 h in mouse monoclonal anti-synapsin (anti- SYNORF1, 1 : 20; Developmental Studies Hybridoma Bank, Iowa City, IA, USA). After rinsing in PBS-Tx, we used an Alexa 546-tagged secondary antibody (1 : 1000; Invitrogen) for detection of antiserum. Finally, the brains were rinsed, dehydrated in an ascending series of ethanol concentrations and cleared and mounted in methyl salicylate. For some of the stages, we also used a rabbit polyclonal antibody against the neuropeptide allatostatin-A (AST-A, 1 : 1000, Agricola) and an Alexa 488-tagged secondary antibody (1 : 1000; Invitrogen) for detection.
10
Immunohistochemical Staining of Insect Brains
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Females were decapitated directly after completion of a treatment. From the decapitated heads, the mouthparts were removed and small holes were pierced in the compound eyes, which was done in phosphate buffer (PB) 0.1 M. The heads were fixed overnight in 4% paraformaldehyde (diluted in PB 0.1 M, pH 7.4). After fixation, heads were washed in PB 0.1 M and phosphate-buffered saline with 2.5% Triton-X (PBS-tx). Brains were dissected in PBS-tx, and thereafter incubated for 72 h with the primary antibody, mouse monoclonal anti-synapsin (anti-SYNORF1; Developmental Studies Hybridoma Bank, Iowa City, IA, USA), diluted 1 : 20 with PBS-tx and 0.5% bovine serum albumin (BSA). Brains were washed with PBS-tx, and then incubated for 48 h with the secondary antibody, anti-mouse Alexa 546 (Invitrogen, Stockholm, Sweden), diluted 1 : 500 with PBS-tx and 0.5% BSA. After staining, the brains were carefully washed first in PBS-tx and then PBS, and subsequently stored at 4°C in PBS with 0.02% sodium azide. Finally, all brains were dehydrated in a series of increasing alcohol concentrations (30–100%), and cleared and stored in methyl salicylate at 4°C.
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