Sc 7819

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Sc-7819 is a laboratory consumable product manufactured by Santa Cruz Biotechnology. It is a tool used for scientific research purposes.

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6 protocols using sc 7819

Fluorescent In Situ Hybridization for AC3 in Brain Sections

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Brain sections (14 μm) were cut using a cryostat and mounted onto Colorfrost Plus slides (Thermo Fisher Scientific). In situ hybridization (ISH) was performed using the RNAscope system (Advanced Cell Diagnostics, Newark, CA, United States) according to the manufacturer’s protocol. Sections were pretreated with hydrogen peroxide for 10 min at RT, followed by target-retrieval solution and Protease III using RNAscope 2.5 Universal Pretreatment Reagents (#322381; Advanced Cell Diagnostics). Then the commercial probe for AC3 (#478071-C1) was used. After hybridization, the RNAscope Multiplex Fluorescent Detection Kit v2 (#323110) was used to amplify signal. After ISH, sections were labeled with anti-SST antibody (goat, 1:500, sc-7819; Santa Cruz), followed by its corresponding secondary antibody (Alexa Fluor 488 donkey anti-goat, 1:200, A-11055; Invitrogen). Images were captured with a confocal laser-scanning microscope (Model FV1000; Olympus).

Yang X.Y., Ma Z.L., Storm D.R., Cao H, & Zhang Y.Q. (2021). Selective ablation of type 3 adenylyl cyclase in somatostatin-positive interneurons produces anxiety- and depression-like behaviors in mice. World Journal of Psychiatry, 11(2), 35-49.

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Immunohistochemistry for Neuronal Markers

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We used the following antibodies: ChAT (Chemicon AB144P), Otx2 (R&D systems goat, catalog # AF1979), Tuj1 (Covance MMS-435P), pH3 (Millipore 06-570), parvalbumin (Millipore MAB1572), somatostatin (Santa Cruz sc-7819).
Cryosections were rinsed in PBS, blocked in 10% normal serum/PBST (1x PBS, 0.1% Triton X-100), incubated in primary antibody overnight (4° C), washed in PBST, incubated in secondary antibody 1–3 hours (room temperature), and washed in PBS. For fluorescent detection, we used Alexa 488- and Alexa 594-conjugated secondary antibodies (Invitrogen). For colorimetric detection, biotinylated secondary antibodies (Vector) were used with the ABC (Vector)/DAB detection method.
For ChAT IHC, antigen retrieval was achieved by incubating slides in 2.94g/L trisodium citrate dehydrate, 0.05% Tween-20, pH 6.0 for 15 minutes at 90° C. Blocking and antibody incubations were done in 1% BSA in PBST. Sections were incubated two days at 4° C with primary antibody, and signal was amplified with biotinylated anti-goat (Vector) prior to fluorescent detection with streptavidin-594 (Invitrogen).
For OTX2 IHC, we modified the IHC protocol according to the recommendations of Yuki Muranishi in the Furakawa lab (Osaka, Japan). Briefly, antigen retrieval was achieved as for ChAT IHC, and samples were blocked in 4% donkey serum in PBST.

Hoch R.V., Lindtner S., Price J.D, & Rubenstein J.L. (2015). OTX2 Transcription Factor Controls Regional Patterning Within The Medial Ganglionic Eminence (MGE) And Regional Identity Of The Septum. Cell reports, 12(3), 482-494.

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Multicolor Immunostaining of Isolated Islets

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Immunostaining was conducted on isolated islets, fixed in 4% paraformaldehyde before being permeabilised using 0.1% Triton X-100 (Sigma). After blocking with 5% goat serum, islets were incubated overnight (4°C) with primary antibodies before incubation with secondary antibodies. Fluorescent staining was visualised using a laser-scanning confocal microscopy (BioRad) controlled by LaserSharp2000 (BioRad).
Antibodies used in this study were: rabbit FITC 495-conjugated anti-GFP (1:250; DS-PB-00926; InsightBio, Wembley, United Kingdom), mouse anti-glucagon (1:1000; G2654; Sigma), guinea-pig anti-insulin (1:400; A0564; Dako, Santa Clara, California, United States), goat anti-somatostatin (1:100; sc-7819; Santa Cruz Biotechnology, Dallas, Texas, United States); Alexa Fluor 633 goat anti-mouse IgG (1:500; A-21052; ThermoFisher); Alexa Fluor 594 goat anti-guinea pig IgG (1:500; A-11076; ThermoFisher); and Alexa Fluor 546 donkey anti-goat IgG (1:100; A-11056; ThermoFisher).

Acreman S., Ma J., Denwood G., Gao R., Tarasov A., Rorsman P, & Zhang Q. (2024). The endoplasmic reticulum plays a key role in α-cell intracellular Ca2+ dynamics and glucose-regulated glucagon secretion in mouse islets. iScience, 27(5), 109665.

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Immunofluorescent Analysis of Hormone Production

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Timing: 4 h

Hormone production is analyzed by immunofluorescent staining of insulin and somatostatin. Hormones will be detected 5 days following exocrine cell isolation.

37.

Aspirate the medium and fix cells for 12 min in 4% PFA

38.

Perform three washes of the cells with PBST (PBS supplemented with 0.2% Triton X-100). In the third wash, leave the cells in PBST for 15 min before continuing with the protocol. All subsequent washes are performed in PBST.

39.

For blocking, incubate the cells for 1 h with CAS-Block.

Optional: supplement CAS-Block with 5% horse serum during blocking.

40.

Dilute primary antibodies in CAS-Block: guinea pig anti-insulin (DAKO, A0564, 1:300) and goat anti-somatostatin (Santa Cruz Biotechnology, SC-7819, 1:200).

41.

Incubate with primary antibodies for 1–3 h at room temperature (20–30°C) or overnight (16–20 h) at 4°C.

42.

Wash the cells three times with PBST

43.

Incubate with secondary antibodies (Jackson ImmunoResearch) diluted 1:200 in CAS-Block for 1 h at room temperature (20–30°C).

44.

Wash the cells three times with PBST

45.

Stain nuclei for 10 min using 1 μg/mL DAPI in DDW.

46.

Wash DAPI with PBS.

47.

If using 24-well plates or ibidi 4-well μ slides, cells can be directly imaged. Alternatively, if using plastic coverslips, mount on slides before imaging.

Elhanani O, & Walker M.D. (2020). Protocol for Studying Reprogramming of Mouse Pancreatic Acinar Cells to β-like Cells. STAR Protocols, 1(2), 100096.

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Immunohistochemistry Protocol for Embryonic and Postnatal Brain Sections

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Immunohistochemistry was performed on 12 μm (embryonic brains) or 30 μm (postnatal brains) coronal sections (Liu et al., 2018 (link)). Briefly, sections were blocked for 30 min in TBS with 0.1% Triton X-100 and 5% donkey serum. For double staining, sections were incubated simultaneously with primary antibodies from different species, and secondary antibodies were used sequentially. Primary antibodies were incubated for 24 h at 4°C. We used rabbit anti-calretinin (CR; 1:3,000, AB5054, Millipore, Burlington, MA, USA), chicken anti-GFP (1:2,000, GFP-1020, Aves Labs), rabbit anti-PV (1:2,000, PV25, Swant), rabbit anti-NKX2-1 (1:500, sc-13040, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-NPY (1:500, 22940, Incstar), goat anti-SST (1:500, sc-7819, Santa Cruz Biotechnology, Dallas, TX, USA) and goat anti-SP8 (1:2,000, sc-104661, Santa Cruz Biotechnology, Dallas, TX, USA). Secondary antibodies (1:400, Jackson Immuno Research) were incubated for 2 h at room temperature (RT), rinsed three times in TBS, and then incubated with DAPI (4′,6-diamidino-2-phenylindole, 1:5,000) for 3 min.

Tao G., Li Z., Wen Y., Song X., Wei S., Du H., Yang Z., Xu Z, & You Y. (2019). Transcription Factors Sp8 and Sp9 Regulate Medial Ganglionic Eminence-Derived Cortical Interneuron Migration. Frontiers in Molecular Neuroscience, 12, 75.

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Quantifying Pancreatic Cell Populations

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Pancreatic sections (5 μm) were deparaffinized and hydrated as described elsewhere (Wierup et al. 2004 (link)). Sections were incubated with primary antibodies against insulin (9003; EuroDiagnostica, Malmö, Sweden), glucagon (7811; EuroDiagnostica), somatostatin (sc-7819; Santa Cruz Biotechnology) and secondary antibodies (Jackson ImmunoResearch). Immunofluorescence was examined in an epifluorescence microscope (Olympus BX60; Olympus), see example hormone staining in Supplementary Fig. 2A. For beta cell mass quantification all islets in three different parts of each pancreas (minimum 200 µm apart) were assessed in a blinded fashion using NIS-Elements software (NIS-Elements 3.1; Nikon). Total insulin-, glucagon-or somatostatin-stained area and total section area were measured and alpha, beta or delta cell mass was calculated as the ratio between the two areas. Beta cell number per islet was calculated using DAPI as a marker of cell nuclei. All nuclei surrounded by insulin staining were regarded as beta cells. An average of 44 ± 4 islets per animal were analyzed (n WT = 21 pancreatic sections from seven mice, n F508del = 21 pancreatic sections from seven mice).

Edlund A., Barghouth M., Huhn M., Abels M., Esguerra J., Mollet I., Svedin E., Wendt A., Renstrom E., Zhang E., Wierup N., Scholte B.J., Flodström-Tullberg M, & Eliasson L. (2019). Defective exocytosis and processing of insulin in a cystic fibrosis mouse model. The Journal of endocrinology.

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