Tissues and cells were lysed with RIPA lysis buffer supplemented with 1% protein phosphatase inhibitor mixture, and the sample was then centrifuged (12,000 rpm) at 4°C for 15 min to get the protein supernatant. The concentration of total protein was detected using the BCA assay kit (Thermo). After separation in a 10–12% SDS-PAGE gel, the proteins were then transferred onto a PVDF membrane (Bio-Rad). The membrane was blocked for 1.5 h with 5% (w/v) non-fat milk dissolved in tris-buffer saline solution containing 0.1% (v/v) Tween-20 (TBST) at RT and further incubated overnight at 4°C with the following primary antibodies: ZO-1 (1:1,000), P120-catenin (1:1,000), β-catenin (1:1,000), occludin (1:1,000), claudin-5 (1:1,000), ATG5 (1:1,000), Beclin1 (1:1,000), AKT (1:1,000), p-AKT (1:1,000), FOXO1 (1:1,000), KLF4 (1:1,000), β-actin (1:10,000), and LC3II (1:1,000; Abcam). Next, after washing three times with TBST, the membrane was submerged in a corresponding HRP-conjugated secondary antibody for 1 h at RT. Signals of protein expression were collected by a ChemiDoc XRS imaging system (Bio-Rad). The relative density of the band was quantified by ImageJ software. All of the primary antibodies (except for anti-LC3II) were purchased from Proteintech.
Anti lc3ii
Manufactured by Proteintech
Sourced in United States
Anti-LC3II is a laboratory equipment product designed for the detection and quantification of the LC3II protein, which is a marker of autophagy. It can be used in various research applications related to the study of autophagic processes.
Automatically generated - may contain errors
Lab products found in correlation
Mouse igg, by Cell Signaling Technology (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Boster Bio (3 mentions)
Cy3 conjugated goat anti mouse secondary antibody, by Wuhan Servicebio Technology (3 mentions)
Fitc conjugated goat anti rabbit secondary antibody, by Wuhan Servicebio Technology (3 mentions)
Rabbit igg, by Cell Signaling Technology (3 mentions)
Fluorescence microscope, by Olympus (2 mentions)
P120 catenin, by Abcam (1 mentions)
Foxo1, by Abcam (1 mentions)
Lc3 2, by Abcam (1 mentions)
Bca assay kit, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
β actin, by Abcam (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
β catenin, by Abcam (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Anti p62, by Novus Biologicals (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Uorescence microscope, by Olympus (1 mentions)
Anti β actin, by Abcam (1 mentions)
Anti nf κb, by Proteintech (1 mentions)
8 protocols using anti lc3ii
1
Protein Expression Analysis Protocol
2
Western Blot Analysis of Neuronal Proteins
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The proteins that were extracted from primary spinal neurons and the sciatic nerve tissues were electrophoresed on 12% SDS-polyacrylamide gel and then transferred to nitrocellulose membranes (Millipore, Carrigtwohill, Ireland), blocked with nonfat milk, and probed with primary antibodies at 4°C overnight; the membranes were washed three times and immunoblotted with secondary antibodies at room temperature for 2 hr. Finally, blots were detected by enhanced chemiluminescence (ECL) (Pierce, Rockford, IL, USA), and protein bands were quantitated with ImageJ software (NIH) using β-actin as an internal control. Primary antibodies were as follows: anti-LC3-II (Proteintech, Chicago, USA), anti-p62 (Novus, Littleton, Colorado, USA), and anti-β-actin (A5441, Sigma-Aldrich Chemicals, St. Louis, MO, USA).
3
Immunofluorescence Analysis of RAW264.7 Macrophages
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RAW264.7 macrophages were washed with PBS, xed with 4% paraformaldehyde and subsequently permeabilized using 0.3% Triton X-100. After blocking with 5% BSA, the cells were incubated with anti-LC3II (1:100, Proteintech Biotechnology, Chicago, USA), or anti-Arg-1 (1:100, Mouse IgG, Cell Signaling Technology, Boston, USA) and anti-iNOS (1:200, Rabbit IgG, Cell Signaling Technology, Boston, USA) antibodies overnight at 4 ℃. Subsequently, cells were rinsed with PBS, then incubated with an FITC conjugated goat anti-rabbit secondary antibody (1:200, Servicebio Biotechnology, Wuhan, China) or Cy3 conjugated goat anti-mouse secondary antibody (1:200, Servicebio Biotechnology, Wuhan, China) and mounted with DAPI (1:200, Boster Biological Technology, China). Finally, the cells were photographed using a uorescence microscope (Olympus Corporation, Japan).
4
Immunofluorescence Staining of Macrophages
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RAW264.7 macrophages were washed with PBS, fixed with 4% paraformaldehyde and subsequently permeabilized using 0.3% Triton X-100. After blocking with 5% BSA, the cells were incubated with anti-LC3II (1:100, Proteintech Biotechnology, Chicago, USA), or anti-Arg-1 (1:100, Mouse IgG, Cell Signaling Technology, Boston, USA) and anti-iNOS (1:200, Rabbit IgG, Cell Signaling Technology, Boston, USA) antibodies overnight at 4 . Subsequently, cells were rinsed with PBS, then incubated with an FITC conjugated goat anti-rabbit secondary antibody (1:200, Servicebio Biotechnology, Wuhan, China) or Cy3 conjugated goat anti-mouse secondary antibody (1:200, Servicebio Biotechnology, Wuhan, China) and mounted with DAPI (1:200, Boster Biological Technology, China). Finally, the cells were photographed using a fluorescence microscope (Olympus Corporation, Japan).
5
Molecular Mechanisms of Bone Homeostasis
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After the soft tissue on the tibia of mice had been removed, tibias were cut by ophthalmic scissors and 80g weighed by electronic scale. The tibia of each sample was placed in the grinding tube, to which had been added grinding beads and lysis buffer, and placed in the tissue homogenizer for grinding. The supernatant and was gotten for examining concentration of total protein by bicinchoninic acid method(BCA). Polyacrylamide gel electrophoresis was performed to separate the target proteins from the samples with the equal amount of total protein. Proteins were transferred to PVDF membrane (Invitrogen) and closed non-specific binding with 5% skimmed milk. The protein was added with primal antibodies of anti-PI3K (1:1000, Abcam), anti-Akt (1:1000, Abcam), anti-pAkt (1:1000, Abcam), anti-mTOR (1:1000, Sigma), anti-Beclin-1 (1:1000, Proteintech), anti-LC3 II (1:1000, Proteintech), anti-p62 (1:1000, Proteintech), anti-NF-κB (1:1000, Proteintech), anti-Runx2 (1:1000, Sigma), anti-beta actin (1:1000, Abcam) and secondary antibodies (Abcam) in turn, and then washed with tris-buffered saline Tween (TBST). The PVDF film was developed and photographed by Alpha gel imaging system. Beta-actin was used as an internal reference, and the data were analyzed by self-contained software.
6
Immunofluorescence Staining of Macrophages
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RAW264.7 macrophages were washed with PBS, fixed with 4% paraformaldehyde and subsequently permeabilized using 0.3% Triton X-100. After blocking with 5% BSA, the cells were incubated with anti-LC3II (1:100, Proteintech Biotechnology, Chicago, USA), or anti-Arg-1 (1:100, Mouse IgG, Cell Signaling Technology, Boston, USA) and anti-iNOS (1:200, Rabbit IgG, Cell Signaling Technology, Boston, USA) antibodies overnight at 4 °C. Subsequently, cells were rinsed with PBS, then incubated with an FITC conjugated goat anti-rabbit secondary antibody (1:200, Servicebio Biotechnology, Wuhan, China) or Cy3 conjugated goat anti-mouse secondary antibody (1:200, Servicebio Biotechnology, Wuhan, China) and mounted with DAPI (1:200, Boster Biological Technology, China). Finally, the cells were photographed using a fluorescence microscope (Olympus Corporation, Japan).
7
Immunohistochemical Analysis of Xenograft Tumors
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The xenograft tumors (control and DSF/Cu groups) were deparaffinized in xylene, dehydrated in graded alcohol, and cut into sections with 4-µm thickness. The sections were deparaffinized, rehydrated, treated with 3% hydrogen peroxide, and blocked with 10% goat serum at 37°C for 30 min. After washing, the sections were incubated with anti-PARP (1:100, Huabio), anticleaved-cas3 (1:100, Huabio), and anti-LC3II (1:100, Proteintech) at 4°C overnight. The sections were then washed with PBS and incubated with appropriate HRP-conjugated secondary antibodies (1:500 dilution, ThermoFisher Scientific, Waltham, MA, USA) at 37°C for 1°h. Finally, the sections were incubated with diaminobenzidine and counterstained with hematoxylin for detection.
8
Hepatoprotective Effects of CGA, APAP, and AG
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CGA (purify ≥ 98%) was purchased from Herbest Co., Ltd. (Baoji, China). APAP was obtained from Shanghai Johnson Pharmaceutical Co., Ltd. (Shanghai, China). AG (purify ≥ 98%) was provided by Abphyto Co., Ltd. (Chengdu, China). Commercial assay kits for alanine transaminase (ALT), aspartate transaminase (AST), and lactate dehydrogenase (LDH) were supplied by Medical System Co., Ltd. (Ningbo, China). A Cell Total RNA Isolation Kit was acquired from Foregene Co., Ltd. (Chengdu, China). Anti-Tom20 was provided by Santa Cruz Biotechnology, Inc. Anti-SQSTM1/p62 and anti-Parkin were offered by Cell Signaling Technology (Danvers, MA, USA). Anti-LC3II, anti-Bcl2 and anti-Bax were provided by Proteintech Group, Inc. (Wuhan, China). Anti-PINK1 was purchased from Abcam (Cambridge, UK). Anti-glyceraldehyde phosphate dehydrogenase (GAPDH), Alexa Fluor® 488 AffiniPure, Goat Anti-Rabbit IgG(H+L) Alexa Fluor®, Alexa Fluor® Cy3 AffiniPure Goat Anti-Mouse IgG(H+L) Alexa Fluor®, Goat Anti-Mouse or anti-Rabbit IgG (H+L) Secondary Antibody HRP, and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) kit were produced by Servicebio (Wuhan, China). The BCA protein assay kit was acquired from Multi Sciences (Hangzhou China).
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