Rabbit anti 5 ht

Manufactured by Merck Group

Sourced in Denmark, United Kingdom, Canada

Rabbit anti-5-HT is a laboratory reagent used for the detection and quantification of the neurotransmitter 5-hydroxytryptamine (5-HT), also known as serotonin, in biological samples. It is a polyclonal antibody raised in rabbits against 5-HT. This product can be used in various immunoassay techniques, such as ELISA, to measure 5-HT levels in research applications.

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10 protocols using rabbit anti 5 ht

Immunostaining of Drosophila CNS

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The CNS was dissected from flies in PBS, and fixed in 4% paraformaldehyde in PBS for 60 min. Immunostaining was carried out as described previously16 (link), using the following antibodies at the indicated dilutions: rabbit anti-GABA diluted at 1:500 (Sigma), rabbit anti-5-HT at 1:500 (Sigma), chicken anti-GFP at 1:1,000 (Abcam), rabbit polyclonal anti-GFP at 1:1,000 (Molecular Probes) and mouse monoclonal nc82 at 1:20 (DSHB, University of Iowa, IA). The secondary antibodies used were as follows: Alexa647-conjugated goat anti-mouse IgG at 1:200, Alexa546-conjugated goat anti-rabbit IgG at 1:200, Alexa488-conjugated goat anti-chicken IgG at 1:200, Alexa488-conjugated goat anti-rabbit IgG at 1:200 and Alexa546-conjugated goat anti-mouse IgG at 1:200 (all from Invitrogen). Stacks of optical sections were obtained with a Zeiss LSM 510 META confocal microscope and were processed with Image J software.

Yilmazer Y.B., Koganezawa M., Sato K., Xu J, & Yamamoto D. (2016). Serotonergic neuronal death and concomitant serotonin deficiency curb copulation ability of Drosophila platonic mutants. Nature Communications, 7, 13792.

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Embryonic and Adult Brain Immunostaining

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Pregnant females were sacrificed by cervical dislocation, the embryos dissected out of the uterus and fixed o/n in 4% paraformaldeyde (PFA) at 4°C. Adult animals were anaesthetized with Avertin and perfused intracardially with 4% PFA. Brains were dissected out, post-fixed o/n in PFA at 4°C and embedded in either 2.5% agarose or tissue-tek for sectioning with vibratome or cryostat, respectively.
For immunostaining specimens were incubated with primary antibodies o/n at 4°C in PBS containing 5% heat-inactivated lamb serum and 0.5% Triton X-100. Primary antibody dilutions: rabbit anti-5-HT (Sigma) 1∶500; chicken anti-eGFP/eYFP (Abcam) 1∶1000; mouse anti-calbindin-D-28K (Sigma), 1∶200. Fluorescent-conjugated secondary antibody were used as follow: Rhodamine Red-X goat anti-rabbit IgG 1∶500; Alexa Fluor 488 goat anti-chicken IgG 1∶200; Rhodamine Red-X goat anti-mouse IgG 1∶500 (all by Molecular Probes). Cell nuclei were counterstained with DAPI (Sigma), 0.5 µg/ml.

Pelosi B., Migliarini S., Pacini G., Pratelli M, & Pasqualetti M. (2014). Generation of Pet1210-Cre Transgenic Mouse Line Reveals Non-Serotonergic Expression Domains of Pet1 Both in CNS and Periphery. PLoS ONE, 9(8), e104318.

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In situ Hybridization and Immunohistochemistry Protocols

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In situ hybridization was performed as previously described in [27 (link)]. Antisense probe for th2 was generated by PCR amplification using the primers below, followed by transcription with T7 RNA Polymerase. th2 primers: 5’- CGGAGACAGCTTCGTGTT, 3’- GCTCATTAGAAAGGGCATA. cfos probe was made as previously described [28 (link)]
Immunohistochemistry was performed as previously described in [18 (link)], with the addition of treatment with 2 N HCl for 30–60 minutes for BrdU detection.
The following primary antibodies were used: Rabbit anti-DA (purchased from H.W.M. Steinbusch), Rabbit anti-5HT (Sigma, S5545), Mouse anti-GFP (Invitrogen, A11120), Rabbit anti-GFP (Invitrogen, A11122), Chicken anti-GFP (Aves, GFP-1020), Rabbit anti-dsRed (Clontech, 632496), Mouse anti-BrdU (DSHB, G3G4-BrdU), Chicken anti-BrdU (Aves, CBDU-65A-Z)
The following secondary antibodies were used: Goat anti-Mouse Alexa Fluor 448 (Invitrogen, A11001), Goat anti-Rabbit Alexa Fluor 488 (Invitrogen, A11008), Donkey anti-Chicken Alexa Fluor 488 (Jackson ImmunoResearch, 703-545-155), Goat anti-Chicken Alexa Fluor 568 (Invitrogen, A11041), Goat anti-Mouse Alexa Fluor 633 (Invitrogen, A21050), Goat anti-Chicken Alexa Fluor 633 (Invitrogen, A21103), Goat anti-Mouse Alexa Fluor 647 (Invitrogen, A21235), Goat anti-Rabbit Alexa Fluor 647 ((Invitrogen, A21244), Goat anti-Chicken Alexa Fluor 647 (Invitrogen, A21449)

McPherson A.D., Barrios J.P., Luks-Morgan S.J., Manfredi J.P., Bonkowsky J.L., Douglass A.D, & Dorsky R.I. (2016). Motor Behavior Mediated by Continuously Generated Dopaminergic Neurons in the Zebrafish Hypothalamus Recovers After Cell Ablation. Current biology : CB, 26(2), 263-269.

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Immunolabeling Protocols for Invertebrate Species

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For staining, nine adult C. fingalianum (Figure 1(a)), five adult C. monotrochum (Figure 2(a)), 12 adult M. striatum (Figure 3(a)), six adult P. koreni (Figure 4(a)), 14 adult A. dioicum (Figure 5(a)), and seven adult V. auriculatum (Figure 6(a)) were used. Cylindrostoma monotrochum and M. striatum were stained according to Grosbusch et al. (2019). Four individuals of V. auriculatum were stored at −20°C before staining; two in ethanol and two in aceton. Furthermore, for A. dioicum, C. fingalianum, P. koreni, and V. auriculatum some modifications of the staining protocol were made. After incubation with the first antibody (rabbit anti‐5HT, Sigma‐Aldrich), animals were washed for 24 h with PBS‐T_x (1× phosphate buffered saline with 0.1% Triton X‐100, Sigma‐Aldrich), and then incubated for 1 h at room temperature (RT) in the secondary fluorescein isothiocyanate‐conjugated (FITC) goat anti‐rabbit antibody (DAKO, Denmark), diluted 1:250 in BSA‐T_x (PBS‐T_x with 1% bovine serum albumin, Carl Roth, Germany). Specimens were washed again with PBS‐T_x for five days at RT and five nights at 4°C in darkness.

Grosbusch A.L., Bertemes P, & Egger B. (2021). The serotonergic nervous system of prolecithophorans shows a closer similarity to fecampiids than to triclads (Platyhelminthes). Journal of Morphology, 282(4), 574-587.

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Immunohistochemical Labeling of Drosophila Brains

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Dissected brains were fixed with 4% paraformaldehyde for 15 min at room temperature. Fixed tissue was blocked with PBS containing 2% Triton X-100 and 10% normal goat serum (NGS) for 30 min and then incubated for 1 day each (with washing in between) at room temperature in PBS containing 1% Triton X-100, 0.25% NGS, and a primary antibody or secondary antibody solution. The brain was mounted in 20 μl Vectashield medium. We used the following primary antibodies at the indicated dilutions: 1:50 mouse anti-bruchpilot (nc82) (Developmental Studies Hybridoma Bank, Iowa), 1:1000 rabbit anti-5HT (Sigma, S5545). Secondary antibodies from Invitrogen were used at dilutions of 1:250, which were Alexa Fluor 633 goat anti-mouse lgG (Life Technologies, A21050), Alexa Fluor 568 goat anti-mouse lgG (Life Technologies, A11004), Alexa Fluor 633 goat anti-rabbit (Invitrogen, A21071). Confocal images were acquired with Zeiss LMS710 confocal laser scanning microscope under 40x or 63x magnification.

Suzuki Y., Schenk J.E., Tan H, & Gaudry Q. (2020). A population of interneurons signals changes in the basal concentration of serotonin and mediates gain control in the Drosophila antennal lobe. Current biology : CB, 30(6), 1110-1118.e4.

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Serotonin Immunostaining in C. elegans and Mouse Embryos

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C. elegans serotonin antibody staining was performed using the tube fixation protocol (McIntire et al., 1992 (link)). Briefly, synchronized young adult hermaphrodites were fixed in 4% paraformaldehyde (PFA) for 18 hr, with β-mercapto-ethanol for another 18 hr, with 1 mg/ml collagenase (Sigma Aldrich, Merk, Darmstadt, Germany) for 90 min and incubated for 24 hr with rabbit anti-5HT antibody (1:5000; Sigma Aldrich). Alexa 555-conjugated donkey anti-rabbit (1:500; Molecular probes) was used as secondary antibody.
For mouse immunohistochemistry, freshly isolated E11.5 embryos from C57Bl/6JRccHsd were fixed by immersion in 4% PFA. Rabbit anti-Sall2 (1:100; Sigma Aldrich), goat anti-Brn2 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-5HT (1:5000; Sigma Aldrich) and goat anti-5HT (1:200; Abcam, Cambridge, UK) antibodies were used. As secondary antibodies Alexa 555-conjugated donkey anti-rabbit and anti-goat, and Alexa 488-conjugated donkey anti-rabbit and anti-goat were used (1:600; Molecular probes, Invitrogen, Eugene, OR). Immunofluorescence samples were analyzed and photographed using a confocal TCS-SP8 Leica microscope.

Lloret-Fernández C., Maicas M., Mora-Martínez C., Artacho A., Jimeno-Martín Á., Chirivella L., Weinberg P, & Flames N. (2018). A transcription factor collective defines the HSN serotonergic neuron regulatory landscape. eLife, 7, e32785.

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Multimarker Immunofluorescence Staining

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Sections from the control group were permeabilized with PBS containing 0.1% Triton X-100 (PBST) and 2% fetal bovine serum and incubated overnight at room temperature with primary antibodies to netrin-G1 (1:500 goat anti-Netrin-G1a, R&D Systems) or p11 (1:100 goat anti-S100A10, R&D Systems) and 5-HT (1:1000 rabbit anti-5HT, Sigma) or p11 and anti-phospho-histone H3 (PH3) (1:200 rabbit anti-phospho S10, Abcam). Slides were then washed in PBST before applying secondary antibodies. Incubation with secondary antibodies (1:800 anti-goat HRP and 1:800 anti-rabbit Rhodamine Red, Jackson ImmunoResearch Laboratories) occurred at room temperature for 2 h in the dark. Slides were again washed in PBST prior to amplification. For amplification, slides were incubated in Tyramide Signal Amplification-Fluorescein solution (1:50; PerkinElmer, NEL701A001 KT) for 10 min at room temperature in the dark. A final series of washes with PBST was performed and the slides were mounted using Prolong Gold with DAPI (to visualize nuclei; Life Technologies, P36931).

King J.R., Velasquez J.C., Torii M, & Bonnin A. (2017). Effect of Maternal ±Citalopram Exposure on P11 Expression and Neurogenesis in the Mouse Fetal Brain. ACS chemical neuroscience, 8(5), 1019-1025.

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Immunohistochemical Analysis of Zebrafish Larvae

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Zebrafish larvae were anesthetized and fixed in 4% paraformaldehyde (PFA) for 2 h at room temperature (RT). The larvae were washed 3 × 30 min in distilled water^{27 (link)} and then were incubated for 1 h in blocking solution (2% normal goat serum, 1% bovine serum albumin, 1%dimethylsulfoxide, 0.1% Triton X-100 in PBS). The larvae were incubated overnight at RT in primary antibodies diluted in blocking solution. The primary antibodies used were mouse anti-GFP (1: 5000 dilution; Invitrogen), rabbit anti-5-HT (1: 4000 dilution; Sigma-Aldrich) and rabbit anti-Desmin (1:20 dilution; Sigma-Aldrich). The larvae were rinsed extensively in PBS with Triton X-100 (PBST) and incubated overnight at RT in secondary antibodies diluted in blocking solution. The secondary antibodies were Alexa Fluor 488 (1: 500 dilution; Life Technologies) or 564 (1: 500 dilution; Invitrogen). After rinsing in PBST, larvae were transferred to 50% glycerol in PBS.

Okamoto S.I, & Hatta K. (2022). Ca2+-imaging and photo-manipulation of the simple gut of zebrafish larvae in vivo. Scientific Reports, 12, 2018.

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Immunohistochemical Characterization of Larval Neural Markers

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Immunohistochemistry was performed essentially as described (Matthews et al., 2007 (link)). Third instar larvae were dissected in 1X PBS, fixed in 4% paraformaldehyde (Electron Microscopy Sciences) in 1X PBS for 15 min, rinsed three times in 1X PBS + 0.3% Triton X-100 (PBS-TX), and blocked for 1 hr at 4°C in normal donkey serum (Jackson Immunoresearch). Primary antibodies used were chicken anti-GFP (1:1000; Abcam), rabbit anti-DsRed (1:250, Clontech), mouse anti-1D4 anti-Fasciclin II (1:10; Developmental Studies Hybridoma Bank), rabbit anti-5HT (1:1000; Sigma), rabbit anti dvGLUT (1:10,000) (Daniels et al., 2004 (link)), rabbit anti-GABA (1:100; Sigma), mouse anti-ChAT (1:100; Developmental Studies Hybridoma Bank). Animals were incubated overnight in primary antibodies at 4°C, rinsed repeatedly in PBS-TX, and incubated overnight at 4°C in species-specific, fluorophore-conjugated secondary antibodies (Jackson ImmunoResearch) at 1:200 in PBS-TX. Tissue was mounted on poly-L-lysine coated coverslips, dehydrated in ethanol series, cleared in xylenes, and mounted in DPX (Fluka).

Burgos A., Honjo K., Ohyama T., Qian C.S., Shin G.J., Gohl D.M., Silies M., Tracey W.D., Zlatic M., Cardona A, & Grueber W.B. (2018). Nociceptive interneurons control modular motor pathways to promote escape behavior in Drosophila. eLife, 7, e26016.

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Immunohistochemical Localization of 5-HT

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Tissues were incubated overnight with rabbit anti-5-HT (1:1000, Sigma-Aldrich, Oakville, ON, Canada) and then visualized with goat anti-rabbit AlexaFluor 488 (1:200, 1h RT, Invitrogen Life Technologies Inc., Burlington, ON, Canada). Slides were coverslipped using Vectashield™ with DAPI (Vector Labs).

Potter L.E., Doolen S., Mifflin K., Tenorio G., Baker G., Taylor B.K, & Kerr B.J. (2018). Antinociceptive effects of the antidepressant phenelzine are mediated by context-dependent inhibition of neuronal responses in the dorsal horn.. Neuroscience, 383, 205-215.

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