Cardiac tissue sections were prepared as previously described. 19 (link) Endothelial cells were stained with rat anti-CD31 antibodies (BD Pharmingen Inc, San Diego, CA, USA) and the presence of Smoothened was assessed with rabbit polyclonal anti-Smo (Abcam, Cat# ab72130). For immunofluorescence microscopy, primary antibodies were resolved with Alexa-Fluor–conjugated secondary antibodies (Invitrogen Corporation, Carlsbad, CA, USA) and nuclei were counterstained with DAPI (1/5000). Slides were imaged using fluorescent microscopy (Zeiss).
Anti smo
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-Smo is a laboratory reagent used for detecting the protein Smoothened (Smo) in various biological samples. Smo is a key component of the Hedgehog signaling pathway, which plays a critical role in embryonic development and tissue homeostasis. Anti-Smo can be used in techniques such as Western blotting, immunohistochemistry, and flow cytometry to identify and quantify Smo expression levels in cells and tissues.
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Lab products found in correlation
Anti gli1, by Abcam (5 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Rat anti cd31 antibody, by BD (2 mentions)
Fluorescent microscopy, by Zeiss (2 mentions)
Anti ptch1, by Merck Group (2 mentions)
Mouse anti β actin monoclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
Anti cd133, by Abcam (1 mentions)
Anti rkip, by Abcam (1 mentions)
Anti acetylated tubulin, by Merck Group (1 mentions)
Secondary antibody staining, by Jackson ImmunoResearch (1 mentions)
Anti histone h3, by Abcam (1 mentions)
Ne per nuclear and cytoplasmic extraction kit, by Thermo Fisher Scientific (1 mentions)
Ecl reagent, by PerkinElmer (1 mentions)
Quantity one 4, by Bio-Rad (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Anti shh, by Abcam (1 mentions)
Anti p camkii, by Cell Signaling Technology (1 mentions)
Anti pcreb, by Cell Signaling Technology (1 mentions)
Anti bax, by Abcam (1 mentions)
11 protocols using anti smo
1
Cardiac Tissue Immunofluorescence Microscopy
2
Western Blot Analysis of Protein Markers
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Proteins were exacted from cells and tissues using RIPA buffer supplemented with the protease inhibitor PMSF. The protein concentrations were measured using the bicinchoninic acid Protein Assay Kit (Beyotime, China). An equal amount of protein in each sample was separated on a sodium dodecyl sulfate–polyacrylamide gel and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat dry milk in PBS for 1 h at room temperature and incubated with primary antibodies at 4°C overnight. After incubation with a secondary antibody, the blots were visualized using enhanced chemiluminescent substrate detection reagent (Beyotime, China). Anti-RKIP, anti-SMO, anti-Gli1 and anti-CD133 (1:1,000) were purchased from Abcam (Cambridge, UK). As a loading control, the expression of β-actin was detected using monoclonal mouse anti-β-actin antibody (1:5,000) from Santa Cruz Biotechnology (CA, USA) according to the manufacturer’s instructions.
3
Immunohistochemical Analysis of NKTCL and LRH
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Paraffin-embedded specimens were obtained from 30 individuals with newly diagnosed NKTCL and 10 individuals with LRH between 2011 and 2018 at the First Hospital of Quanzhou Affiliated to Fujian Medical University. The sampled were subjected to IHC staining assay utilizing the following polyclonal antibodies: anti-Smo (1: 100) and anti-Gli1 (1: 150) (Abcam, Cambridge, UK). We then counted all tumor cells and Smo or Gli1 staining tumor cells using a microscope (×400) in 4 fields. The percentage score was determined by the average percentage of tumor cells with positive staining: 0 (<10%), 1 (11–20%), 2 (21–50%), 3 (51–75%), and 4 (>76%). The intensity score depended on the staining intensity: 0 (negative), 1 (weak brown), 2 (brown), and 3 (strong brown). The level of Smo or Gli1 expression was obtained by multiplying percentage score and intensity score, divided into 4 groups: negative (−) (0), positive (+) (1–3), positive (++) (4–7), and positive (+++) (8–12).
4
Cardiac Tissue Immunofluorescence Microscopy
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Cardiac tissue sections were prepared as previously described. 19 (link) Endothelial cells were stained with rat anti-CD31 antibodies (BD Pharmingen Inc, San Diego, CA, USA) and the presence of Smoothened was assessed with rabbit polyclonal anti-Smo (Abcam, Cat# ab72130). For immunofluorescence microscopy, primary antibodies were resolved with Alexa-Fluor–conjugated secondary antibodies (Invitrogen Corporation, Carlsbad, CA, USA) and nuclei were counterstained with DAPI (1/5000). Slides were imaged using fluorescent microscopy (Zeiss).
5
Quantifying Ciliary Smoothened Localization
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Fibroblasts were plated on glass coverslips. Following stimulation with Shh-N cells were fixed for 1 h with 4% PFA then permeabilized for 10 min with 0.5% Triton X-100 in PBS, followed by blocking in 1% bovine serum albumin, 0.1% Triton X-100 and overnight incubation with anti-Smo (Abcam) and anti acetylated-tubulin (Sigma). Following secondary antibody staining (Jackson) coverslips were mounted and then imaged with a Leica SP5. Six confocal planes 0.5 μm apart containing ∼200 cells per field were imaged. ImageJ was used for image analysis: the maximum intensity of each pixel across all confocal planes was determined by maximum intensity projection of acquired z-stacks; Acetylated-tubulin staining was used to automatically segment cilia; mean SMO immunostaining per cilia pixel was calculated by dividing the sum of the maximum intensities of each cilium-assigned pixel by the area of the cilium; the value of a manually assigned background pixel was subtracted from all plotted data.
6
Shh Signaling Pathway Temporal Expression
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To identify temporal expression of Shh signaling (Shh, Ptch1, Smo, Gli1), whole-cell protein extract lysates were used. To identify the activation of Shh signaling, nuclear extracts were prepared using an NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce Biotechnology) according to the manufacturer’s instruction. L4-L5 spinal cord segments were quickly removed from deeply anesthetized rats and homogenized in ice-cold RIPA lysis buffer containing a cocktail of protease inhibitors. Total proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to 0.2 µm polyvinylidene difluoride membrane. The following primary antibodies were used: anti-Shh (1:2000, Abcam), anti-Ptch1(1:1000, Sigma), anti-Smo (1:2000, Abcam), anti-Gli1 (1:2000, Abcam), anti-p-GluN2B (Tyr1472) (1:500, Millipore), anti-p-CaMKII (1:1000, Cell signaling Tech), anti-p-CREB (1:1000, Cell signaling Tech), anti-Histone H3 (1:1000, Abcam), and anti-GAPDH (1:10,000, Sigma). The filters were developed using ECL reagents (Perkin Elmer) with secondary antibodies from Millipore Bioscience Research Reagents. Data were analyzed with a Molecular Imager (Gel DocTM XR, 170–8170) and the associated software Quantity One-4.6.5 (Bio-Rad Laboratories).
7
Western Blot Analysis of Cell Lysates
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Anti-cTnT, anti-β-actin and anti-Ki67 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-GATA4 and anti-desmin were bought from Cell Signaling Technology (Danvers, MA, USA). Anti-SMO, anti-Bax and anti-Bcl-2 antibodies were purchased from Abcam (Cambridge, MA, USA). Cells were added into tubes containing lysis buffer (50 mmol/l Tris-HCl, 0.2% sodium deoxycholate, 0.2% SDS, 1% Triton X-100, and 1 mmol/l EDTA at pH 7.4). The lysate supernatant was collected after centrifugation at 14,000 g for 30 min at 4°C. Subsequently, protein concentrations were determined using a BCA protein detection kit (Keygen Biotech. Co. Ltd., Nanjing, China) according to the manufacturer's instructions. Western blot assay was performed as previously described (Shen et al., 2012 (link)).
8
Western Blot Analysis of Hedgehog and BDNF Signaling
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Proteins were extracted from the ipsilateral T4-5 spinal cord dorsal horn (SDH), and the supernatant protein concentrations were detected with the BCA protein assay kit (Abcam). The equal number of sample proteins was separated on sodium dodecyl sulphate–polyacrylamide gels. Subsequently, they were transferred onto the polyvinylidene fluoride membrane, which was blocked with 5% non-fat milk and then incubated with primary antibodies overnight at 4°C. The primary antibodies included anti-Shh (1:2000, GeneTex, San Antonio, USA), anti-Ptch1 (1:1000, Sigma, St. Louis, USA), anti-Smo (1:2000, Abcam), anti-Gli1 (1:2000, Abcam), anti-BDNF (1:1000, GeneTex), anti-p-TrkB (1:1000, GeneTex), anti-TrkB (1:2000, GeneTex), anti-p-PI3K (1:1000, GeneTex), anti-p-Akt (1:1000, GeneTex), anti-Histone H3 (1:2000, GeneTex), and anti-GAPDH (1:10,000, GeneTex). Finally, the membranes were rinsed and the proteins were detected with the enhanced chemiluminescence method.
9
Inhibition of Sonic Hedgehog Pathway
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Anti-Smo, anti-Gli1, and anti-MGMT antibodies were purchased from Abcam (Cambridge, UK). Anti-Cyclin D1 and anti-CDK-6 antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). The anti-β-actin antibody was purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA), and GANT61, GDC0449, and lomeguatrib were obtained from Selleck Chemicals (Houston, TX, USA). The TMZ was purchased from J&K Scientific (Beijing, People’s Republic of China).
10
Western Blot Analysis of Polyamine Metabolism
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Cells were washed with PBS and lysed in RIPA buffer (Nacalai Tesque) containing 10 µg/ml aprotinin, 500 µM sodium orthovanidate and 10 µg/ml phenylmethylsulfonyl uoride. Proteins were separated on a 10% SDS-polyacrylamide gel and transferred electrophoretically to Immobilon-E transfer membranes (Merck Millipore). Blots were blocked in 5% nonfat dry milk in tris-buffered saline containing 0.1% Tween 20 (TBS-T) for 30 min at room temperature. The levels of speci c proteins were detected with an ECL Western Blotting Detection System (GE Healthcare) using anti-ODC1 (Proteintech), anti-AMD1 (Cell Signaling Technology), anti-spermidine synthase (Proteintech), anti-spermine synthase (Proteintech), anti-SMO (Abcam), anti-AcPAO (Abcam), anti-SSAT1 (Abcam), anti-protein conjugated acrolein (PC-Acro, Takara Bio, Japan) and anti-actin (Santa Cruz Biotechnology). Images were captured using an Amersham Imager 600 (GE Healthcare) and bands were quanti ed using the ImageJ program (Schneider et al. 2012 ), normalized to actin and expressed as relative amount.
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