For immunohistochemical detection of insulin granules, paraffin-embedded sections of the pancreas were deparaffinized in xylene, rehydrated in descending grades of alcohol and then immersed in 0.3% hydrogen peroxide (Sigma-Aldrich) for 30 min to inactivate endogenous peroxidase activity. The samples were heated in 10 mM citrate buffer (Sigma-Aldrich) at 121°C for 30 min for antigen retrieval, blocked in 5% normal serum (Sigma-Aldrich) for 20 min, and incubated with a rabbit polyclonal anti-insulin [dilution, 1:100 in phosphate-buffered saline (PBS); sc-9168; Santa Cruz Biotechnology Inc., Dallas, TX, USA] antibody overnight at 4°C. Subsequently, three PBS washes were performed and the sections were incubated with a goat anti-rabbit biotin-conjugated secondary antibody (dilution, 1:2,000 in PBS; sc-2040; Santa Cruz Biotechnology, Inc.) for 20 min at room temperature. After further incubation with horseradish peroxidase-labeled streptavidin (Vector Laboratories, Inc., Burlingame, CA, USA), the antibody binding was visualized with diaminobenzidine (Sigma-Aldrich) and sections were counterstained with hematoxylin. Negative controls, in which the primary or secondary antibodies, or the avidin-biotin complex reagent was omitted, produced no positive staining. Positive controls were used according to the instructions provided by the manufacturer's of the primary antibodies.
Sc 9168
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-9168 is a lab equipment product offered by Santa Cruz Biotechnology. It serves a core function, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 goat anti rabbit antibody, by Thermo Fisher Scientific (4 mentions)
Flag tag (flag), by Merck Group (3 mentions)
Sc 2040, by Santa Cruz Biotechnology (2 mentions)
Orca er ccd camera, by Hamamatsu Photonics (2 mentions)
Axio observer z1 inverted microscope, by Zeiss (2 mentions)
Impress anti rabbit hrp, by Vector Laboratories (2 mentions)
Axiovision software, by Zeiss (2 mentions)
Novared substrate, by Vector Laboratories (2 mentions)
Insulin l6b10, by Cell Signaling Technology (2 mentions)
Sc 374565, by Santa Cruz Biotechnology (2 mentions)
Ab13520 50, by Abcam (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Spa 827, by Stressgen (2 mentions)
Gm130, by BD (2 mentions)
Normal serum, by Merck Group (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
10 mm citrate buffer, by Merck Group (1 mentions)
Ab932, by Merck Group (1 mentions)
Dmrxa2 microscope, by Leica (1 mentions)
19 protocols using sc 9168
1
Immunohistochemical Insulin Granule Detection
2
Quantifying Pancreatic Beta Cell Area
3
Quantifying Pancreatic Beta Cell Area
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Pancreata were isolated from control and STZ-treated dams after delivery of pups, and from neonatal male and female rats on postnatal day 1. Specimens were fixed in formalin overnight, embedded in paraffin and sectioned by the Histology Core of the Department of Anatomy and Cell Biology at the Indiana University School of Medicine. Staining for insulin was performed (sc-9168, 1:500, Santa Cruz Biotechnology, Dallas, TX), with detection using Impress anti-rabbit HRP and Novared substrate (Vector Laboratories, Burlingame, CA). A Zeiss Axio Observer Z1 inverted microscope (Zeiss, Thornwood, NY) equipped with an Orca ER CCD camera (Hamamatsu Photonics, Hamamatsu City, Japan) was used to acquire digital images of the entire stained longitudinal pancreatic section. The beta cell area of at least 5 sections, each separated by at least 75 μm, from at least 3 animals in each group was calculated using Axio-Vision Software (Zeiss, Thornwood, NY) as previously described (48 (link)).
4
Immunohistochemical Staining of Pancreatic Islets
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After overnight fixation in 4% paraformaldehyde at 4 °C, the pancreases dissected from neonatal mice were putted in 10% sucrose for 4 h, 20% sucrose for 8 h, and 30% sucrose for 12 h, sequentially. The pancreases slices were first incubated at 4 °C with blocking buffer for 90 min, and then incubated overnight at 4 °C with anti-insulin (cat. #sc-9168, Santa Cruz, 1:250), anti-SST (cat. ab30788, Abcam, 1:500), or anti-glucagon (cat. #AB932, Millipore, 1:250) antibodies. After washing three times with PBS, the slices were incubated at 4 °C for 2.5 h with goat anti-rabbit antibody (cat. #A27034, Invitrogen) or goat anti-rat antibody (cat. #ab150158, Abcam). The slices were stained with 4′, 6′-diamidino-2-phenylindole and subjected to fluorescence microscopic analysis as previously described47 (link),48 (link).
5
Histological Analysis of Pancreatic Tissue
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Frozen sections were prepared by fixing tissues overnight in 4 % paraformaldehyde, followed by cryoprotection in 30 % sucrose at 4 °C for two days and sectioning with given thickness for histological and immunofluorescence analysis. For morphological analysis, Section (5 μm) were stained with hematoxylin and eosin to have images acquired with a Leica DMRXA2 microscope. For immunofluorescence analysis, Section (6–8 μm) were treated following the standard protocol with following antibodies: rabbit anti-OTG1 (Sigma HPA018019, 1:1000), Alexa 488 conjugated rabbit-anti-Giantin (Covance A488-114L, 1:1000), rabbit anti-insulin (Santa Cruz sc-9168, 1:1000), goat anti-glucagon (Santa Cruz sc-7780, 1:1000), donkey anti-goat IgG-FITC (Chemicon AP180F, 1:2000), donkey anti-rabbit IgG-Cy3 (Millipore AP182C, 1:2000).
6
Immunohistochemical Analysis of Insulin Expression
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To examine the expression of insulin in pancreatic tissues, an immunohistochemical examination was carried out with insulin antibody (SC-9168; Santa Cruz Biotechnology, Heidelberg, Germany). The 5 μm thick paraffin sections were deparaffinized in xylene and hydrated with ethanol. Then the hydrated sections were treated with 3% H2O2 in methanol for 30 min to block any endogenous peroxidase. The sections were washed at least three times with 0.01M phosphate buffer (pH 7.4) for 10 min; and processed further by an indirect immunoperoxidase technique using a One-Step Polymer-HRP Detection kit (Leica Biosystems, Newcastle, United Kingdom) with secondary antibodies. The sections were counterstained with hematoxylin. The stained sections were observed under microscope and representative images captured.
7
Immunohistochemical Analysis of Rat Pancreas
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Rat Pancreas was fixed in 10% buffered neutral formalin then washed, dehydrated in ascending grades of ethyl alcohol then cleared in xylene, embedded in paraffin, sectioned at 5 micron thickness, deparaffinized, and then treated with 3% H2O2 for 10 min to block the peroxidases activity. Subsequently, the tissue samples were heated at 121°C in 10 mM citrate buffer for 30 min, blocked for 20 min in 5% normal serum. The pancreatic tissue was incubated overnight with rabbit polyclonal anti-insulin primary antibody (1:100; sc-9168; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or with GLUT4 Antibody (1:100; sc-53566; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) in phosphate-buffered saline (PBS) at 4°C. After three extensive washes with PBS, the sections were incubated with a goat anti-rabbit IgG biotin conjugated secondary antibody (1:2,000; sc-2040; Santa Cruz Biotechnology, Inc.) for 20 min at 32°C. After further incubation with horseradish peroxidase-labeled streptavidin, antibody binding was visualized using diaminobenzidine, and the sections were counterstained with hematoxylin.
8
Immunohistochemical Analysis of Pancreatic Markers
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Western blotting, immunostainings and β-galactosidase were performed as previously described[8 (link)]. Antibodies for indicated antigens include beta-catenin 1:200 in PBS (AB6302, Abcam), insulin 1:800 in PBS (sc-9168, Santa cruz), glucagon 1:1200 in PBS (PA039-5P, BioGenex), e-cadherin 1:500 in MOM diluent (610182, BD Bioscience), beta-galactosidase 1:1000 in PBS (8559762, MP Biomedical), and amylase 1:5000 in Tris Buffered saline (Sc-46657, Santa Cruz). Reagents: DAPI (5mg/mL) 1:4000 in PBS (D1306, Thermofisher) and 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside 1mg/mL in PBS (Sigma-Aldrich, B4252-100MG) and mouse on mouse (MOM) basic kit (BMK-2202, Vector Laboratories) according to manufacturer’s descriptions, ABC basic kit 1:25 (PK-4000, Vector Laboratories) and 3–3’-Diaminobenzene (DAB) (SK-4100, Vector Laboratories) in the presence of 0.0005% (v/v) hydrogen peroxide (H2O2).
9
Antibody Sourcing for Protein Research
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The following antibodies were purchased: β-actin (sc-47778, Santa Cruz Biotechnology, Santa Cruz, CA, USA), insulin (L6B10, Cell Signaling Technology, Danvers, MA, USA, and sc-9168, Santa Cruz Biotechnology), UGGT1 (14,170–1-AP, Proteintech, Rosemont, IL, USA; sc-374565, Santa Cruz Biotechnology; and ab13520-50, Abcam), and FLAG (F-1804, Sigma, St. Louis, MO, USA). UGP knockdown siRNA (sc-154894) was purchased from Santa Cruz31 (link).
10
Immunofluorescence Staining of NIT-1 Cells
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The following antibodies were purchased: insulin (L6B10, Cell Signaling Technology, Danvers, MA, USA and sc-9168, Santa Cruz, California CA, USA), GM130 (cis-Golgi network staining, 610822, BD Transduction Laboratories, New York, NY, USA), FLAG (F-1804, Sigma, St Louis, MO, USA), HA (anti-HA high affinity 3F10, Roche, Basel, Switzerland), KDEL (ER staining, SPA-827, Stressgen, Victoria, BC, Canada), and Alexa Fluor-conjugated secondary antibodies (Thermo Fisher, Rockford, IL, USA). Mouse pancreas-derived NIT-1 cells (purchased from ATCC® CRL-2055TM, Manassas, VA, USA) were maintained as described previously18 (link). Briefly, the cells were plated at a density of 1.5–3.0 × 106 cells/60-mm dish, and the medium (F-12K, Kaighn’s Modification of Ham’s F-12 Medium, 2 mmol/l L-arginine and 7 mmol/l glucose) after 48 h of culture medium was exchanged to fresh medium after 48 h of culture.
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