Western blot was performed as previously described [22 (link)]. Total proteins (30 ug) from lung tissue were separated by SDS-PAGE gels and then transferred to 0.22-µm PVDF membranes (Bio-Rad). After blocking, the membranes were incubated with primary antibodies against RhoA (1:1000 dilution; cat. no. 2117s; CST), ROCK1 (1:1000 dilution; ab156284; Abcam), MLC2 (1:1000 dilution; cat. no. 3672s; CST), p-MLC2 (1:1000 dilution; cat. no. 3671s; CST), 8-OHdG (1:500 dilution; sc-393,871; Santa Cruz), or α-Tubulin (1:1000 dilution; cat. no. 3873 S; CST) overnight at 4˚C, respectively; followed by the appropriate horseradish peroxidase-conjugated secondary antibodies (Dako). The expression was visualized using an enhanced chemiluminescence detection kit (Thermo Scientific). Semiquantitative analysis was done using ImageQuant LAS 4000 mini (GE Healthcare Life Sciences). Additional file 1 is the original WB image in the manuscript.
Sc 393871
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-393871 is a laboratory equipment product offered by Santa Cruz Biotechnology. It is designed for use in research and scientific applications. The core function of this product is to facilitate specific laboratory procedures and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Ab201340, by Abcam (2 mentions)
Fitc conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
A11017, by Thermo Fisher Scientific (2 mentions)
Laser scanning confocal microscope, by Nikon (2 mentions)
Enhanced chemiluminescence detection kit, by Thermo Fisher Scientific (1 mentions)
0.22 m pvdf membrane, by Bio-Rad (1 mentions)
Imagequant las 4000 mini, by GE Healthcare (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Agilent Technologies (1 mentions)
Ab23751, by Abcam (1 mentions)
Alexa fluor 488 donkey anti mouse igg, by Abcam (1 mentions)
Alexa fluor 555 donkey anti rabbit igg, by Abcam (1 mentions)
Af1817, by R&D Systems (1 mentions)
Ab75941, by Abcam (1 mentions)
Ab209484, by Abcam (1 mentions)
Cm1950, by Leica camera (1 mentions)
Cy3 labeled goat anti rabbit igg, by Beyotime (1 mentions)
Alexa fluor 488 labeled goat anti rabbit igg, by Beyotime (1 mentions)
7 protocols using sc 393871
1
Western Blot Analysis of Lung Tissue
2
Immunofluorescent Analysis of 8-OHdG and Fibronectin
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Formalin-fixed, paraffin-embedded monkey TM sections were used for immunochemistry staining. After deparaffinizing and antigen retrieval, two antibodies were applied to the same specimen. A mouse monoclonal antibody of 8-OHdG, (sc-393871; Santa Cruz Biotechnology, Dallas, TX, USA) was used at 1:500 dilution, and a rabbit polyclonal antibody of fibronectin (ab23751; Abcam, Cambridge, MA, USA) was used at 1:500 dilution at 4°C overnight, respectively. Alexa-Fluor 488 donkey anti-mouse IgG and Alexa-Fluor 555 donkey anti-rabbit IgG (1/1000; Abcam) were used as secondary antibodies to detect 8-OHdG and fibronectin separately. 4′,6-Diamidino-2-Phenylindole (DAPI) was used for nuclear staining. ImageJ (http://imagej.nih.gov/ij/ ; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA) was used for fluorescence intensity analysis.
3
Immunofluorescence Analysis of Oxidative Stress
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After fixation (4% paraformaldehyde for 10 min), permeabilization (0.3% Triton X-100 for 10 min), and blocking (1% BSA for 1 h), cells or sections were incubated with mouse anti-8-OHdG (sc-393871, Santa Cruz Biotechnology, Dallas, TX, USA), anti-mouse KIM-1 (AF1817, R&D Systems, Minneapolis, MN, USA), anti-mouse CD68 (ab201340, Abcam) and anti-human ACE2 (266699–1-Ig, Proteintech) overnight at 4 °C. After washing with PBS, slides were incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibody (A11017, Life Technologies, CA, USA) at 37 °C for 1 h. Nuclei were visualized by staining with DAPI. Digital images were captured using a confocal laser scanning microscope (Nikon).
4
Oxidative Stress in Retinal Pigment Epithelium
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Aβ1-40/PBS-treated mouse RPE-choroid complex and incubated primary RPE cells were fixed with 4% paraformaldehyde. The analysis was performed as previously described 7 (link). Primary antibodies against the following were used for staining: 8-OHdG (1:100, sc-393871, Santa Cruz), cytochrome b245 light chain/p22phox (1:500, ab75941, Abcam), and NOX4 (1:500, PA5-72816, Thermo Fisher Scientific).
5
Immunohistochemical Analysis of AMPK and Oxidative DNA Damage
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For prepared decalcified sections, IHC staining was processed with primary antibodies against phospho-AMPK (CST, 2535 T, 1:100 dilution) and 8-OHdG (8-hydroxy-20-deoxyguanosine, a marker of DNA damage in oxidative stress, Santa Cruz Biotechnology, sc-393871, 1:200 dilution) as previously reported [32 (link), 44 (link)]. For quantitative valuation, three slices from each group were observed using an optical microscope and analyzed by Image-Pro Plus 6.0. The mean integrated optical density (IOD) for pAMPK and 8-OHdG were also calculated around the implants.
6
Nano-indentation and Immunofluorescence for Femur Slices
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Following the nano-indentation, immunofluorescence staining of 50 μm thick proximal femur slices was performed as previously described [61 (link)]. Briefly, after treatment with 0.3% Triton (9002-93-1, Solarbio, Beijing, China) for 1.5 h, slices were blocked with 4% Bovine serum albumin (BSA) at room temperature for 2 h and incubated overnight at 4 °C with primary antibodies: Osterix (ab209484, Abcam, Cambridge, UK, 1:400), TRAP (bs-16578R, Bioss, Beijing, China, 1:800), and 8-OHdG (sc-393871, Santa Cruz, CA, USA, 1:50). Then, secondary antibodies were incubated at room temperature for 2 h to visualize primary antibodies: Cy3-labeled Goat Anti-Rabbit IgG (A0516, Beyotime, Shangahi, China, 1:400) and Alexa Fluor 488-labeled Goat Anti-Rabbit IgG (A0423, Beyotime, China, 1:400). Finally, the slices were mounted with mounting media containing DAPI (36308ES20, Yeasen, Shangahi, China). A laser confocal microscope (CM1950, Leica, Wetzlar, Germany) was used for observation. Positive cells per unit area in the growth plate, and positive cells per unit length around trabeculae and cortical bone were calculated by Image J software (Figure 1 (Fiv)).
7
Immunofluorescence Staining of Cellular Markers
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After fixation (4% paraformaldehyde for 10 min), permeabilization (0.3% Triton X-100 for 10 min), and blocking (1% BSA for 1 h), cells or sections were incubated with mouse anti-8-OHdG (sc-393871, Santa Cruz Biotechnology, Dallas, TX, USA), anti-mouse KIM-1 (AF1817, R&D Systems, Minneapolis, MN, USA), anti-mouse CD68 (ab201340, Abcam) and anti-human ACE2 (266699-1-Ig, Proteintech) overnight at 4 °C. After washing with PBS, slides were incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibody (A11017, Life Technologies, CA, USA) at (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted March 14, 2022. ; https://doi.org/10.1101/2022.03.13.484180 doi: bioRxiv preprint 37 °C for 1 h. Nuclei were visualized by staining with DAPI. Digital images were captured using a confocal laser scanning microscope (Nikon).
The copyright holder for this preprint this version posted March 14, 2022. ; https://doi.org/10.1101/2022.03.13.484180 doi: bioRxiv preprint 37 °C for 1 h. Nuclei were visualized by staining with DAPI. Digital images were captured using a confocal laser scanning microscope (Nikon).
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