Protein expression in aortic sections of vitamin D3-administered mice was assessed using fluorescent immunohistochemistry as previously described23 (link) using anti-Adam19 antibody (NBP1-69364; Novus Biologicals) and anti-smooth muscle actin antibody (sc-53015; Santa Cruz Biotechnology) with a 1:100 dilution. The photographs of aortic sections were visualized using a Zeiss LSM 710 confocal microscope (Carl Zeiss).
Sc 53015
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-53015 is a laboratory product offered by Santa Cruz Biotechnology. It is a technical-grade reagent, the core function of which is to serve as a research tool for scientific investigations.
Automatically generated - may contain errors
Lab products found in correlation
Ab139645, by Abcam (2 mentions)
Ab124332, by Abcam (2 mentions)
Vectashield fluorescent mounting medium, by Vector Laboratories (2 mentions)
C2 confocal laser scanning microscope system, by Nikon (2 mentions)
Hoechst 33342, by Merck Group (2 mentions)
Cytofix cytoperm, by BD (2 mentions)
Lsm 710 confocal microscope, by Zeiss (1 mentions)
Chemidoctm imaging system, by Bio-Rad (1 mentions)
Enhanced chemiluminescence detection system, by Santa Cruz Biotechnology (1 mentions)
Sc 8422, by Santa Cruz Biotechnology (1 mentions)
Sc 47724, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Shandon cryomatrix, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 donkey anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Ab185617, by Abcam (1 mentions)
Chemiluminescence system, by Merck Group (1 mentions)
Sc 47778, by Santa Cruz Biotechnology (1 mentions)
Ab47405, by Abcam (1 mentions)
Ab254268, by Abcam (1 mentions)
10 protocols using sc 53015
1
Immunohistochemical Analysis of Aortic Protein Expression in Vitamin D3-Treated Mice
2
Protein Expression Analysis in Lung Tissue
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Lung tissues were homogenized with a Polytron homogenizer. Protein extracts were lysed by protein lysis buffer and quantified using the Bicinchonic acid protein assay. Samples (20 μg) were separated by Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (8–15%) gel and then transferred to polyvinylidene fluoride membranes. Nonspecific binding proteins were blocked with 5% skim milk for 1 h at RT. The membranes were incubated with primary antibodies against anti-α-SMA (SC-53015; Santa Cruz Biotechnology, TX, USA) and anti-JAM2 (ab139645; Abcam, Cambridge, UK) overnight at 4 °C. The chemiluminescence signal was scanned using the ChemiDOCTM imaging system (Bio-Rad Laboratories, Hercules, CA, USA).
3
Evaluating Protein Levels in Pancreatic Tissue
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Western blotting was performed to evaluate the protein expression levels of α-SMA and fibronectin. Pancreatic tissue homogenates (120 μg protein/lane) were separated by 8% to 12% SDS-PAGE and transferred onto nitrocellulose membranes (Amersham Inc., Arlington Heights, IL, USA) by electroblotting. Membranes were then incubated with antibodies against α-SMA (1:5,000, sc-53015; Santa Cruz Biotechnology, Dallas, TX, USA), fibronectin (1:100, sc-8422; Santa Cruz Biotechnology), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:3,000, sc-47724; Santa Cruz Biotechnology) at 4°C overnight. Following incubation, membranes were washed with TBS with Tween-20. The primary antibodies were then detected using horseradish peroxidase (HRP)-conjugated secondary antibodies, and visualized using enhanced chemiluminescence detection system (Santa Cruz Biotechnology) according to the manufacturer’s instruction. GAPDH was used as loading control. Expression levels were standardized to GAPDH expression level. Further, data were processed and quantified using volume analysis and molecular analysis software, respectively.
4
Immunofluorescence Analysis of Intestinal Tissues
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Human colon biopsies and mice bowel samples were embedded into Shandon cryomatrix (Thermo Fisher Scientific) and cut into 5 μm slides, stored at -80 °C until use. HT-29 and CCD-18Co cells were seeded in chambers and cultured for 24 h in 37 °C. After repeated washing slides were permeabilized with Cytofix/Cytoperm (BD) for 15 min at RT, then incubated with primary antibodies specific to αSMA (sc-53015; mouse, 1:2000, Santa Cruz Biotechnology, Dallas, TX, USA) or IL-20RB (ab124332; rabbit, 1:100, Abcam) for 1 h at RT. After repeated washing slides were incubated with Alexa Fluor 488 goat anti-mouse IgG (A11001, Invitrogen) and Alexa Fluor 568 donkey anti-rabbit secondary antibody (A10042, Invitrogen), both diluted to 1:100 for 30 min at RT in the dark and counterstained with Hoechst 33,342 (1:2000, Sigma-Aldrich). Finally, slides were rinsed in PBS and coverslipped with Vectashield fluorescent mounting medium (Vector Laboratories, Burlingame, CA, USA). Appropriate controls were performed omitting the primary antibodies to assure their specificity and to avoid autofluorescence. Sections were analyzed with a Nikon C2 confocal laser scanning microscope system (Nikon, Minato, Tokyo, Japan).
5
Western Blot Analysis of Protein Expression
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Total proteins were extracted from the tissues using RIPA lysis buffer as previously described [44 (link)]. Cell lysates were resolved on 10% sodium dodecyl sulfate polyacrylamide gels and transferred to PVDF membranes. The membranes were blocked with 5% skim milk and incubated with primary antibodies overnight at 4 °C, which included an anti-phospho Src antibody (ab185617, Abcam, Cambridge, UK, 1:1000 dilution), anti-Src antibody (ab47405, Abcam, 1:600 dilution), anti-TGF-β1 antibody (sc-146, Santa Cruz Biotechnology, Dallas, TX, USA, 1:400 dilution), anti-fibrinogen antibody (sc-18029, Santa Cruz Biotechnology, 1:800 dilution), anti-α-SMA antibody (sc-53015, Santa Cruz Biotechnology, 1:800 dilution), and anti-β-actin antibody (sc-47778, Santa Cruz Biotechnology, 1:1000 dilution). The membranes were washed and incubated with the secondary antibodies for 2 h at room temperature. Protein expression was detected using a chemiluminescence system (Millipore, Billerica, MA, USA) according to the manufacturer’s specifications.
6
Immunohistochemical Analysis of Arterial Tissues
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Arterial sections from the vitamin D3-treated mice were analyzed for protein expression by fluorescent immunohistochemistry with anti-ATF3 antibody (ab254268, Abcam) and anti-α-smooth muscle actin antibody (sc-53015, Santa Cruz Biotechnology) as described previously.15 (link) The image was acquired using a Leica DM3000 microscope with a Nikon DS-Ri2 camera and Nikon NIS-Elements AR software (Nikon Instruments Korea, Seoul, Korea).
7
Immunofluorescence Labeling of α-SMA, NF-κB, and MCP-1 in Cells
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For immunofluorescence double labelling, the sections were incubated with a mouse monoclonal anti‐α‐SMA antibody (sc‐53015; Santa Cruz, 1:300) and a rabbit anti‐NF‐κB p65 antibody (sc‐109; Santa Cruz, 1:200) or a rabbit anti‐MCP‐1 antibody (bs1101R; Bioss, 1:200) for 48h at 4°C. Antimouse DyLight 594 (BA1141; Boster, 1:500) and anti‐rabbit DyLight 488 (BA1127; Boster, 1:400) fluorescein secondary antibodies were incubated for 1 hour. Finally, the nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI, Roche) for 5 minutes. Images were captured with a laser scanning confocal microscope (Olympus FV3000).
In vitro, PSCs cultured on 20 × 20‐mm slides were fixed in 4% paraformaldehyde, permeabilized in 0.5% Triton X‐100 (Solarbio Life Sciences Co., Ltd), blocked (normal goat serum and 0.5% Triton X‐100) and incubated with a rabbit anti‐p65 antibody or anti‐α‐SMA antibody at 4°C overnight. The cells were incubated for 1 hour with goat anti‐rabbit fluorescein isothiocyanate secondary antibody, and the nuclei were counterstained with DAPI. Images were captured with a ZEISS Imager A2 Fluorescence Microscope (Carl Zeiss AG).
In vitro, PSCs cultured on 20 × 20‐mm slides were fixed in 4% paraformaldehyde, permeabilized in 0.5% Triton X‐100 (Solarbio Life Sciences Co., Ltd), blocked (normal goat serum and 0.5% Triton X‐100) and incubated with a rabbit anti‐p65 antibody or anti‐α‐SMA antibody at 4°C overnight. The cells were incubated for 1 hour with goat anti‐rabbit fluorescein isothiocyanate secondary antibody, and the nuclei were counterstained with DAPI. Images were captured with a ZEISS Imager A2 Fluorescence Microscope (Carl Zeiss AG).
8
Immunohistochemical Analysis of Corpus Cavernosum
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Corpus cavernosum tissues were fixed overnight in 4% paraformaldehyde. Paraffin-embedded tissue specimens were routinely prepared and sectioned at a 5 μm thickness. After antigen repair, goat serum was applied at RT for 30 min. The caspase1 antibody (1:50; 22915-1-AP, Proteintech), NLRP3 antibody (1:50; 19771-1-AP, Proteintech), α-SMA antibody (1:100; SC-53015, Santa Cruz), and CD31 antibody (1: 100; ab222783, Abcam) were incubated with the sample overnight, and goat anti-rabbit IgG (HRP) (Abcam, ab205718) was added through a drip. We used DAPI to stain the cell nuclei, and images were captured using a laser confocal microscope (Nikon, Tokyo, Japan).
9
Immunofluorescence Staining of IL-20RB and α-SMA
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FHs74Int and MFs were seeded in chambers and cultured for 24 h in 37 °C. After repeated washing slides were permeabilized with Cytofix/Cytoperm (BD Pharmingen, San Diego, CA, USA) for 15 min at RT, then washed again, and incubated with primary antibody specific for IL-20RB (ab124332; rabbit, 1:100, Abcam) or α-SMA (sc-53015; mouse, 1:1000, Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at RT. Thereafter the slides were washed with WashPerm solution and incubated with the corresponding Alexa Fluor 568 or 488 conjugated secondary antibody (1:200 anti-rabbit or anti-mouse, Invitrogen) for 30 min (min) at RT in the dark and counterstained with Hoechst 33342 (1:2000, Sigma-Aldrich). Finally, slides were coverslipped with Vectashield fluorescent mounting medium (Vector Laboratories). Appropriate controls were performed by omitting the primary antibodies to assure their specificity and to avoid autofluorescence. Sections were analyzed with a Nikon C2 confocal laser scanning microscope system (Nikon Corporation, Tokyo, Japan).
10
Immunofluorescence Staining of Lung Tissues
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For immunofluorescence staining, 4-μm-thick lung sections were dewaxed with xylene and rehydrated with a gradient of ethanol. The sections were subjected to antigen retrieval with a citrate buffer bath (pH 6) at boiling temperature and then blocked for endogenous peroxidase activity. After washing with phosphate-buffered saline (PBS), the sections were incubated with primary antibodies against alpha smooth muscle actin (α-SMA) (SC-53015; Santa Cruz Biotechnology, Dallas, TX, USA), JAM2 (ab139645; Abcam, Cambridge, UK), Neutrophil (Neu) (ab2557; Abcam, Cambridge, UK), Macrophage (Mac) (ab56297; Abcam, Cambridge, UK), and dendritic cell (DC) (NB110-85474; Novus Biologicals, Toronto, ON, Canada) overnight at 4 °C. The sections were rinsed with PBS and incubated with secondary FITC- or RFP-labelled antibodies for 30 min. Nuclei were counterstained with 4′,6-diamidino-2-phenylidone (DAPI) and the fluorescence images were captured with a fluorescence microscope (IX-51, Olympus, Japan).
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