For immunofluorescence assays, cells were fixed with 4% paraformaldehyde for 30 minutes, followed by permeabilization using 0.5% Triton X-100 for 10 minutes. Next, cells were blocked with 5% bovine serum albumin for 1 hour and incubated with primary antibodies against Runx2 (1 : 200, Cell Signaling Technology), OPA1 (1 : 200, Cell Signaling Technology), cleaved caspase 3 (1 : 200, Cell Signaling Technology), LC3II (1 : 200, Cell Signaling Technology), or p-AMPK (1 : 200, Cell Signaling Technology) overnight at 4°C. The next day, cells were incubated with an appropriate secondary antibody (1 : 200, Cell Signaling Technology) for 1 hour at 37°C. Images were acquired using a fluorescence microscope (Olympus DX51, Tokyo, Japan). Apoptosis was detected using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (Roche, Basel, Switzerland) according to the manufacturer's instructions. The apoptosis index was calculated by calculating the percentage of TUNEL-positive cells to total nucleated cells stained by DAPI.
Lc3 2
Manufactured by Cell Signaling Technology
Sourced in United States, China
LC3-II is a protein found in cells that is involved in the process of autophagy, which is the degradation and recycling of cellular components. It serves as a marker for the formation of autophagosomes, which are double-membrane structures that engulf cellular material and transport it to the lysosome for degradation. The amount of LC3-II present in a cell can be used to measure the level of autophagy activity.
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Lab products found in correlation
Beclin 1, by Cell Signaling Technology (30 mentions)
Cleaved caspase 3, by Cell Signaling Technology (16 mentions)
Lc3 1, by Cell Signaling Technology (16 mentions)
β actin, by Cell Signaling Technology (15 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (14 mentions)
Bcl 2, by Cell Signaling Technology (13 mentions)
Caspase 3, by Cell Signaling Technology (13 mentions)
Gapdh, by Cell Signaling Technology (11 mentions)
P mtor, by Cell Signaling Technology (8 mentions)
β actin, by Merck Group (7 mentions)
Caspase 9, by Cell Signaling Technology (6 mentions)
P akt, by Cell Signaling Technology (6 mentions)
Cleaved parp, by Cell Signaling Technology (6 mentions)
P jnk, by Cell Signaling Technology (5 mentions)
P erk, by Cell Signaling Technology (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
P ampk, by Cell Signaling Technology (4 mentions)
Cytochrome c, by Cell Signaling Technology (4 mentions)
Bcl xl, by Cell Signaling Technology (4 mentions)
Phospho mtor, by Cell Signaling Technology (4 mentions)
102 protocols using lc3 2
1
Immunofluorescence Assay for Apoptosis Markers
2
Western Blot Analysis of Protein Biomarkers
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For each sample, all the cells were lysed for 30 min in lyses buffer on 4°C; after ultrasound, the cell debris was centrifuged at 12,000 rpm for 20 min at 4°C, protein concentration was detected by BCA (Beyotime, Haimen, China), using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to separate the proteins, the separated proteins were blotted onto PVDF membrane, blocking 2 h at room temperature with 5% nonfat dry milk in TBST, incubating the antibody for 16 h at 4°C with 1:1,000 concentration, washing PVDF membrane 3 times for 10 min in TBST, incubating secondary antibody at 25°C for 2 h with the 1:10,000 concentration, washing membranes 3 times for 10 min in TBST. Finally, the membranes were visualized with the enhanced chemiluminescence (ECL) detection system (GE Healthcare, Piscataway, NJ, USA). All of the primary antibodies (p-JNK, JNK, P62, LC3-II, GAPDH) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).
3
Spinal Cord Injury: Immunofluorescent Analysis
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Rats were deeply anesthetized with chloral hydrate 12 h after operation (n=3), and transcardiac perfusion was performed as previously described (43 (link)). A 2-cm-long spinal cord segment centered on the injured area was removed and placed into 4% paraformaldehyde overnight, and then samples were cryoprotected in 30% sucrose buffer for cryosectioning. The spinal cords were sectioned into 5-µm-thick coronal sections with a cryostat and used for immunofluorescent staining. Each section was washed with phosphate-buffered saline (PBS) and treated with 0.1% Triton X-100 for 5 min. Sections were then blocked with 10% bovine serum albumin (BSA) for 1 h, then incubated with primary antibody at 4°C overnight as follows: MBP (1:150), Galc (1:100), GFAP (1:100) (Cell Signaling Technology, Inc.). After washing with PBS for 1 h, sections were incubated with the appropriate fluorescent-labeled goat-anti rabbit secondary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and then examined under a fluorescence microscope (Nikon, Tokyo, Japan). Specimens were collected from rats killed at 12 h, 1 week and 2 weeks postoperatively and stained with LC3-II (1:50; Cell Signaling Technology, Inc.), and then observed under a fluorescence microscope (Nikon).
4
Comprehensive Cell Signaling Antibody Protocol
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Antibodies against phospho-Histone H3, TOPK, Histone H3, PAPR, caspase 3 were obtained from “Cell Signaling Technology” (USA), β-actin and horseradish peroxidase (HRP) conjugated secondary antibody from rabbit and mouse were purchased from “Santa Cruz” (USA) and “Protein Tech Group” (USA), respectively. The chemiluminescence's detection kit ECL Plus was from “GE Healthcare” (USA). The Dulbecco's Modified Eagle medium (DMEM), Roswell Park Memorial Institute medium (RPMI 1640), phosphate buffered saline (PBS), trypsin, fetal bovine serum (FBS) were purchased from Thermo Scientific. Hoechst 33342 was purchased from Sigma-Aldrich (St. Louis, MO, USA). All other common chemicals, solvents and reagents were of highest grade available from various commercial sources.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), MDC, Rhodamine 123, and Hoechst 33258 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against PARP, Bcl-xL, Bax, cytochrome c, caspase-8, caspase-9, β-actin, p62, LC3-II, Beclin-1, p-p38, p-38, p-JNK, JNK, p-Erk, Erk, Histone H3 and NF-κB were purchased from Cell Signaling Technology (CST, Beverly, MA). NF-κB SN50 was from Merck Millipore (USA).
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), MDC, Rhodamine 123, and Hoechst 33258 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against PARP, Bcl-xL, Bax, cytochrome c, caspase-8, caspase-9, β-actin, p62, LC3-II, Beclin-1, p-p38, p-38, p-JNK, JNK, p-Erk, Erk, Histone H3 and NF-κB were purchased from Cell Signaling Technology (CST, Beverly, MA). NF-κB SN50 was from Merck Millipore (USA).
5
Preparation and Analysis of RA-XII
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RA-XII (purity ≥ 99%) was prepared in our laboratory, which was identified by MS and NMR spectroscopies [27 (link)]. RA-XII was dissolved at the concentration of 20 mM in DMSO as a stock solution, stored at −20 °C, and diluted with medium for each experiment. DMEM, fetal bovine serum and lipofectamine 2000 were purchased from Life Technologies (Burlington, ON, Canada). Chloroquine (Sigma, St. Louis, MO, USA) was dissolved in phosphate-buffered saline. Rapamycin (Sigma, St. Louis, MO, USA), Z-VAD-FMK (Sigma, St. Louis, MO, USA), MDC (Sigma, St. Louis, MO, USA) and AO (Sigma, St. Louis, MO, USA) were dissolved in DMSO (<0.1%). GFP-LC3B plasmid was purchased from Miaolingbio (Wuhan, China). The antibodies as follows: LC3-II, ATG3, 12, 16, Bcl-2, p-AMPK, p-Akt, p-mTOR, and p-P70S6K (Cell Signaling Technology, Danvers, MA, USA); and GAPDH, Akt, AMPK, mTOR, P70S6K, caspase 3, 8, 9 and PARP (Proteintech, Chicago, IL, USA).
6
Autophagy Quantification by Western Blot
7
Immunofluorescence Analysis of Kidney Samples
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Normal human adult kidney samples were collected from kidneys nephrectomised due to renal cancer (Karolinska University Hospital, Stockholm, Sweden). Nephrin and podocin antibodies have been described previously9 (link),23 (link). Other antibodies were: Med22—Atlas Antibodies and Sigma; Vimentin, alpha SMA, WT-1—Sigma (human); CD31—Abcam; Clathrin, Caveolin, Lc3II, Atg16L, Atg5, Beclin, Rab5, Rab7, Rab11 (Cell Signalling); Synaptopodin, Rab3b and LAMP2 (Santa Cruz), mouse nephrin (Acris), mouse WT1 (Millipore).
The samples were snap-frozen, and the cryosections (10 μm) were post-fixed with cold acetone (− 20 °C) followed by blocking in 5% normal goat serum. The primary antibodies were incubated overnight at 4 °C, followed by a 1-h incubation with the secondary antibody. For double-labelling experiments, the incubations were performed sequentially.
The samples were snap-frozen, and the cryosections (10 μm) were post-fixed with cold acetone (− 20 °C) followed by blocking in 5% normal goat serum. The primary antibodies were incubated overnight at 4 °C, followed by a 1-h incubation with the secondary antibody. For double-labelling experiments, the incubations were performed sequentially.
8
Immunoblotting Analysis of Signaling Proteins
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Equal volumes of total protein lysates were separated on a denaturing polyacrylamide gel, which were then processed for wet transfer onto a nitrocellulose membrane followed by probing of the blots with antibodies to detect phospho-p70 S6 kinase (Thr421/Ser424), phospho-Akt (Ser473), phospho-mTOR (Ser2448), phospho-PKC, total p70 S6 kinase, total Akt, total mTOR, and LC3-II (Cell Signaling Technology) and a compatible horseradish peroxidase (HRP)-conjugated secondary antibody for enhanced chemiluminescence-based detection. To normalize for variations in the protein loading on individual lanes, the blots were stripped and re-probed with an antibody against α-tubulin (Accurate Chemical and Scientific Corporation, Westbury, NY, USA).
9
Protein Extraction and Western Blot Analysis in Rat Hearts
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The membrane and mitochondrial protein were extracted immediately after the rats' hearts were isolated, according to the operating instructions of Membrane Protein Extraction Kit (Boster, Wuhan, China) and Mitochondria Isolation Kit (Boster) respectively. The total proteins were extracted from the frozen ventricle tissues by RIPA. Total 40 μg samples were fractionated by SDS-PAGE and transferred to Immobilon-P membranes (Millipore, Billerica, MA, USA) as previously described [1 (link)], and then done with primary antibodies against Cx43, Bcl-2, Bcl-xL, Bax, AKT, phosphorylation of AKT (p-AKT), active Caspase-3, Beclin-1, LC3-II (1:1000, rabbit species, all from Cell Signal Technology) and survivin (1:1000, goat species, Santa Cruz Biotechnology, SantaCruz, CA, USA) at 4°C overnight. Rinsed three times in TBS, the filters blots were incubated with HRP-conjugated rabbit or goat second antibodies (1:5000; Kang Chen Biotechnology, Guangzhou, China) for 1 hr at room temperature. The bands were visualized by an ECL Western-blotting Detection Reagents (Catalogue RPN2106; GE Healthcare, Fairfield, CT, USA) with LAS-3000 detect system. To quantify the immunobloted bands, optic density was analysed using ImagePro 5.0 (Media Cybernetics Inc., Silver Spring, MD, USA).
10
Immunohistochemical Analysis of Cartilage Tissues
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The isolated cartilage tissues were fixed in 4% neutral formalin for 24 h, embedded in paraffin and serially sectioned at 5 µm. Further, the sections were deparaffinized and rehydrated, then submerged in hydrogen peroxide to quench peroxidase activity following incubated with 1% BSA to block non-specific binding sites. Afterward, the primary antibodies were incubated at 4°C for 12 h, and then secondary antibodies were applied for another 1 h at room temperature. All the sections were visualized using diaminobenzidine (DAB, Beyotime) under a light microscope (Leica Microsystems, Wetzlar, Germany). Images were taken at 200× magnification and the scale bar = 50 μm. Antibodies in immunohistochemical analysis were purchased Cell Signaling Technology (Beverly, MA, U.S.A.), including LC3-II (#3868) and P62 (#23214).
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