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4 protocols using ab189955

1

Immunofluorescence Analysis of SCUBE3 and TGFβIIR

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Bel7404 and HepG2 cells were plated in 35 mm confocal culture dishes for 24 h. On the following day, the cells were treated as previously described [24 (link)]. Primary antibodies against SCUBE3 (ab189955, Abcam,1:50) and TGFβIIR (sc-17799, Santa cruz,1:50) were used at 1:50 dilution. After washing, the cells were then incubated with fluorescence-labelled secondary antibodies Alexa-Fluor 555 anti-mouse IgG and anti-rabbit IgG (1:200 dilution, Cell Signaling Technology, Danvers, MA, USA). Then, cells were washed and stained with DAPI. Finally, images of cells positive for SCUBE3 and TGFβIIR were captured using a confocal microscope (Nikon Corporation, Tokyo, Japan).
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2

Immunohistochemical Analysis of SCUBE3

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Immunohistochemical analysis was conducted according to a standard protocol. Briefly, samples were incubated with anti-SCUBE3 (ab189955; Abcam) and anti-mitochondria (ab92824; Abcam) as primary antibodies, followed by washing and incubation with horseradish peroxidase-conjugated secondary antibodies. Histometric observations were performed as described for the histological assessment.
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3

Western Blot Analysis of Dental Proteins

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Total cell proteins were lysed using radioimmunoprecipitation assay (Beyotime) according to the manufacturer’s protocol. Proteins in the conditioned medium were extracted using a liquid sample total protein extraction kit (Solarbio, Beijing, China). Western blotting was performed as previously described [10 (link)]. The primary antibodies used were anti-SCUBE3 (ab189955; Abcam), anti-AMBN (orb155652; Biorbyt, Cambridge, UK), TGFβR1 (ab31013; Abcam), anti-DSPP (sc-73632; Santa Cruz), anti-DMP1 (ab103203; Abcam), anti-OPN (ab8448; Abcam), anti-OSX (ab13418; Abcam), anti-BMP2 (ab14933; Abcam), anti-BMPR1A (ab264043; Abcam), anti-p-SMAD1/5 (9516; Cell Signalling Technology, Boston, MA, USA), and anti-SMAD1 (D59D7; Cell Signalling Technology). Anti-GAPDH (Rayantibody, Beijing, China) was used as an internal control. Antibody binding was detected using an enhanced chemiluminescence kit (Millipore, Billerica, MA, USA). The intensity of the bands was quantified using the ImageJ software (NIH, USA).
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4

Immunohistochemical Detection of SCUBE3

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TMA slides were depara nized and rehydrated through graded alcohols and then subjected to an antigen retrieval procedure (10 mM sodium citrate, 0.05% Tween-20, pH 6.0 for 25 min). Endogenous peroxidase was blocked with 0.3% hydrogen peroxide for 10 min at room temperature. After incubation with the primary rabbit polyclonal anti-SCUBE3 antibody (ab189955, 1:200 dilution, Abcam, Cambridge, UK) overnight at 4 °C, immunoreactivity was developed with an Envision System (DakoCytomation, Glostrup, Denmark) with diaminobenzidine as chromogen. The slides were nally counterstained with hematoxylin (Zymed Laboratories Inc., San Francisco, CA, USA). Incubation with a normal rabbit IgG instead of the primary antibody was not immunoreactive (data not was shown), supporting a good speci city anti-SCUBE3 antibody.
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