Rat anti sst

Manufactured by Merck Group

Sourced in United States

Rat anti-SST is a laboratory reagent used in research applications. It is an antibody that specifically binds to somatostatin (SST), a regulatory peptide hormone. The core function of this product is to enable the detection and study of somatostatin in biological samples.

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8 protocols using rat anti sst

Embryonic and Postnatal Brain Immunohistochemistry

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Pregnant females were euthanized by lethal intraperitoneal (i.p.) injection of pentobarbital (50 mg kg⁻¹), embryos were collected by caesarian cut and brains were dissected and fixed ON in cold 4% paraformaldehyde (PFA) dissolved in 0.1 M phosphate buffer, pH 7.4. For postnatal brains, animals were deeply anaesthetized by i.p. injection of pentobarbital, transcardially perfused with 0.9% saline followed by cold 4% PFA and postfixed (ON) in cold 4% PFA. Brains were cut on a Vibratome (Leica, VT1000S) for IHC and for free-floating in situ hybridization. Sections were kept at 4 °C in 0.1 M phosphate buffer saline and were stained by IHC as described31 (link) with the following primary antibodies rabbit anti-GFP (1:500; Millipore), goat anti-GFP (1:1,000; Chemicon), mouse anti-human ovalbumin upstream promoter transcription factor 2 (COUP-TFII; 1:500; Perseus proteomics), goat anti-SP8 (1:50; Santa-Cruz), rabbit anti-NKX2.1 (1:100; Santa-Cruz), mouse anti-PV (1:1,000; Swant), rat anti- SST (1:500; Millipore), mouse anti-Reelin (1:500; Abcam), rabbit anti-VIP (1:500; Abcam), rabbit anti-NPY (1:1,000; Immunostar), rabbit anti-Calretinin (1:1,000; Swant). Secondary goat or donkey Alexa-488, -568 and -647 antibodies (Molecular Probes, Invitrogen) raised against the appropriate species were used at a dilution of 1:500–1,000 and sections were counterstained with Hoechst 33258 (1:10,000).

Murthy S., Niquille M., Hurni N., Limoni G., Frazer S., Chameau P., van Hooft J.A., Vitalis T, & Dayer A. (2014). Serotonin receptor 3A controls interneuron migration into the neocortex. Nature Communications, 5, 5524.

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Immunostaining Protocol for Cultured Cells

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Immunostaining for cultured cells was carried out as described previously (Yamamizu et al. 2013 (link)). Briefly, 4% paraformaldehydefixed cells were blocked by 1% skim milk (BioLab) and incubated overnight with primary antibodies at 4°C. For immunofluorescent staining, anti-mouse, rat, rabbit, or goat IgG antibodies conjugated with Alexa488 or Alexa546 (Invitrogen) were used as secondary antibodies. Primary antibodies were as follows: mouse anti-TUJ1 (1:500; Covance, Princeton, NJ); rabbit anti-TUJ1 (1:500; Covance); mouse anti-MAP2 (1:500; Sigma-Aldrich, St. Louis, MO); mouse anti-NEUN (1:100; Millipore); rabbit anti-TH (1:500; Millipore); rat anti-DAT (1:500; Millipore); rabbit anti-VMAT2 (1:500; Millipore); rabbit anti-AADC (1:500; Abcam); rabbit anti-ALDH1A1 (1:500; Abcam); rabbit anti-GABA (1:500; Sigma-Aldrich); mouse anti-GAD67 (1:500; Millipore); mouse anti-PV (1:500; Millipore); rat anti-SST (1:500; Millipore); mouse anti-ISL1/ISL2 (1:500; DSHB); mouse anti-Hb9 (1:500; DSHB); goat anti-ChAT (1:500; Sigma-Aldrich). Differentiated neurons were photographed with inverted fluorescent microscopy (Eclipse TE300; Nikon) with the use of NIS-Elements Software (Nikon).

Teratani-Ota Y., Yamamizu K., Piao Y., Sharova L., Amano M., Yu H., Schlessinger D., Ko M.S, & Sharov A.A. (2016). Induction of specific neuron types by overexpression of single transcription factors. In vitro cellular & developmental biology. Animal, 52(9), 961-973.

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Immunohistochemistry of Fixed Brain Tissue

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Brains were fixed by transcardiac perfusion followed by 1 hr of postfixation on ice with 4% formaldehyde/PBS solution. Brains were rinsed with PBS and cryoprotected by using 30% sucrose/PBS solution overnight at 4°C. Cryosections were prepared at 20 μm (for counting immunoprofiles) thickness. Immunohistochemistry was performed by using a PBS solution containing 1.5% normal goat serum and 0.25% Triton X-100 for all procedures. The washing steps were done with PBS. The sections were incubated overnight (ON) at 4°C with selected antibodies, followed by incubation at 4°C ON with donkey secondary antibodies (Jackson ImmunoResearch Laboratories).
For cell counting and post hoc examination of marker expression, sections were stained using rat anti-GFP, mouse anti-Reelin (1:500, MBL, D223-3), rabbit anti-dsRed (Abcam, ab62341), rabbit anti-VIP (1:1,000, Immunostar, 20077), Gt-PV (1:500, Swant:PVG214), and rat anti-SST (1:500, Millipore). For the analysis of nuclear localization of NFATc4 protein, rabbit NFATc4 (Abcam, ab62613, 1:500) was used.

Priya R., Paredes M.F., Karayannis T., Yusuf N., Liu X., Jaglin X., Graef I., Alvarez-Buylla A, & Fishell G. (2018). Activity Regulates Cell Death within Cortical Interneurons through a Calcineurin-Dependent Mechanism. Cell reports, 22(7), 1695-1709.

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Immunohistochemistry for Neuron Subtypes

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Slices containing biocytin-filled cells were fixed overnight in a buffered solution containing 4% PFA. Slices were rinsed in PBS, then blocked and permeabilized in PBS with 5% Donkey Serum, 0.3% Triton X-100 and 1% BSA. Slices were immuno-stained overnight with one or two primary antibodies: rabbit anti-PV (Swant; diluted 1:200), rat anti-SST (Millipore, diluted 1:200), or rabbit anti-VIP (Immunostar, diluted 1:200). Slices were washed 6 × 10min in PBS containing 0.3% Triton X-100. Slices were incubated with donkey anti-rabbit Alexa-488, donkey anti-rat Alexa 594 secondary antibody (1:800, Thermo Fisher), and Streptavidin-647 (1:300, Thermo Fisher) overnight at 4°C. After washing 6 × 10min in PBS with 0.3% Triton X-100, slices were mounted with an aqueous mounting medium. Confocal mages were obtained as described above.

Alexander D.A., Klein S., Gray N.M., Dewar I.G, & Eagles J.M. (2000). Suicide by patients: questionnaire study of its effect on consultant psychiatrists. BMJ : British Medical Journal, 320(7249), 1571-1574.

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Mapping Cortical Interneurons via CTb Tracing

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5–7 days after CTb injection, mice were transcardially perfused with PBS followed by 4% PFA solution, and brains were post-fixed for at least one day. Coronal sections (75 mm) were obtained using a vibratome, and immuhistochemistry was performed (as described above). The following primary antibodies were used to stain for IN markers: rabbit anti-PV (Swant; diluted 1:200); rat anti-SST (Millipore, diluted 1:200); rabbit anti-VIP (Immunostar, diluted 1:200); rabbit anti-NPY (Immunostar, diluted 1:500); rabbit anti-calretinin (Immunostar, diluted 1:500); rabbit anti-nNOS (Life technologies, diluted 1:500), and goat anti-CTb (List, diluted 1:500). The following secondary antibodies were used: donkey anti-rabbit Alexa 488; donkey anti-rat Alexa 488; and donkey anti-goat Alexa 594. For each IN marker, confocal images collected from mounted sections were used to manually count the number of CTb+ and IN marker+ PFC neurons (ImageJ software).

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Immunofluorescent Labeling of Neuronal Markers

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Immunofluorescent labeling was performed on 25 µm cryo-sectioned tissue with the following antibodies: mouse anti-Kv3.1b 1:400 (NeuroMab cat. # 75-041); rabbit anti-phosphoS6^SER240/244 1:400 (Cell Signaling Technologies cat. # 5364); rabbit anti-PV 1:400 (Swant cat. # PV 27); rat anti-SST 1:200 (Millipore cat. # MAB354). The appropriate 488, 594 or 647 Alexa-conjugated secondary antibodies were from Thermo Fisher Scientific (1:300 dilution). Sections were cover-slipped with Vectashield (Vector labs).

Malik R., Pai E.L., Rubin A.N., Stafford A.M., Angara K., Minasi P., Rubenstein J.L., Sohal V.S, & Vogt D. (2019). Tsc1 represses parvalbumin expression and fast-spiking properties in somatostatin lineage cortical interneurons. Nature Communications, 10, 4994.

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Immunohistochemistry Protocol for Neural Markers

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The procedures for immunohistochemistry were carefully performed under the same conditions to evaluate differences between the groups as described in previous studies [9 (link),25 (link)]. The brain sections were rehydrated in graded concentrations of alcohol and quenched endogenous peroxidase activity with 0.3% hydrogen peroxide (H₂O₂) in phos-phate-buffered saline (PBS). For retrieving antigens in tissues, the brain sections were heated in citrate buffer (pH 6.0) with the retriever (2100 Antigen Retriever, Prestige medical, Lancashire, UK). Additionally, the slides were then cooled at 25 °C and washed in PBS. For protein blocking, the sections were incubated in 5% normal goat serum for 30 min. For primary antibody binding, the tissues were incubated in diluted rat anti-SST (1:100, EMD Millipore, Temecula, CA, USA), rabbit anti-Ki67 (1∶500; Abcam, Cambridge, UK, a marker for proliferating cells), or rabbit anti-doublecortin (DCX, Abcam, a marker for developing neuroblasts) antibodies for 48 h at 4 °C. Thereafter, they were exposed to biotinylated goat anti-rat IgG or goat anti-rabbit IgG (1:200, Vector Laboratories, Burlingame, CA, USA), streptavidin peroxidase complex (Vector Laboratories, Burlingame, CA, USA), and then visualized with 3,3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich) in 0.1 M Tris-HCl buffer (pH 7.4).

Yoo D.Y., Kim W., Jung H.Y, & Hwang I.K. (2023). Postnatal Changes of Somatostatin Expression in Hippocampi of C57BL/6 Mice; Modulation of Neuroblast Differentiation in the Hippocampus. Veterinary Sciences, 10(2), 81.

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Immunofluorescence Staining of Brain Sections

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Sections were transferred from antifreeze solution to PBS and washed 3× in PBS. Heat-induced epitope retrieval was then performed by incubating sections in 10 mM sodium citrate (pH 8.5) at 80°C for 30 min. Sections were then incubated at room temperature in a blocking/permeabilization buffer consisting of 2% w/v nonfat-dried milk, 0.3% Triton X, and 0.01% sodium azide in PBS. Sections were incubated in primary antibodies diluted in 2% normal donkey serum and 0.02% Tween20 in PBS for 24–72 h at 4°C. Primary antibodies used in this study: guinea pig anti-PV (1:750, Immunostar), rat anti-SST (1:300, EMD Millipore), rabbit anti-active caspase 3 (1:1000, Sigma-Aldrich), rabbit anti-serotonin transporter (1:1000, Immunostar). Sections were incubated in secondary antibodies diluted 1:1000 in PBS for 2 h at room temperature. All secondary antibodies were Alexa Fluor-conjugated affinity-purified IgG raised in donkey host (Jackson). Sections were incubated in 300 nM 4′,6-diamidino-2-phenylindole (DAPI) diluted in PBS for 15 min at room temperature. Sections were mounted on SuperFrost Plus slides (Fisher Scientific) using Fluoromount-G (Southern Biotech) and #1.5 cover slips.

Gallerani N, & Au E. (2020). Loss of Clustered Protocadherin Diversity Alters the Spatial Distribution of Cortical Interneurons in Mice. Cerebral Cortex Communications, 1(1), tgaa089.

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