After 72 h in 4% cis-butylene solution, all specimens were fixed and decalcified in 10% ethylenediaminetetraacetic acid (EDTA) and stored for 25 days at 4 °C. After the calcium was eliminated, the skull was dehydrated, cleaned, prepared in paraffin blocks in accordance with standard histological procedures, and sliced at 5 μm. Dyeing of histological Sects. (8 μm) for the target of interest was performed. Hematoxylin and eosin (H&E) staining were performed thereafter. For BMP2, LC3, p-ULK1, and ULK1 immunochemical staining, the sections were stained with the following primary antibodies: anti-LC3 (1:500; Ab Cam USA), anti-BMP2 (1:500; Ab Cam USA), anti-ULK1 (1:500, Ab Cam USA), and antip-ULK1 (1:500; CST USA). Peroxidase assays were performed using the Vectastain ABC kit with DAB substrate kit. ImageJ was used to quantify the area of newly formed bone tissue.
Anti ulk1
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-ULK1 is a protein that plays a key role in the regulation of autophagy, a cellular process that involves the degradation and recycling of damaged or unnecessary components. This product is a research tool used to study the function and localization of ULK1 in various biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Anti lc3, by Abcam (3 mentions)
Anti bmp2, by Abcam (1 mentions)
Anti beclin1, by GeneTex (1 mentions)
Anti lc3, by GeneTex (1 mentions)
Ampkα, by Santa Cruz Biotechnology (1 mentions)
Anti beclin 1, by Santa Cruz Biotechnology (1 mentions)
Anti nmdar2b, by Abcam (1 mentions)
Anti lc3a b, by Abcam (1 mentions)
Anti nmdar1, by Abcam (1 mentions)
Beclin 1, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Anti p62, by Abcam (1 mentions)
Chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Chemidoc touch system, by Bio-Rad (1 mentions)
Anti beclin1, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Radio immunoprecipitation assay ripa buffer, by Beyotime (1 mentions)
5 protocols using anti ulk1
1
Bone Formation Dynamics: Histological Analysis
2
Protein Quantification in Autophagy
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Primary antibodies anti-Beclin1 and anti-LC3 (catalogue numbers: GTX113039 and GTX48634 respectively; GeneTex Inc., Irvine, CA), anti-ULK1 (catalogue number: EPR4885(2); Abcam, Cambridge, UK), and anti-Atg7 (catalogue number: DF6130; Affinity Biologicals Inc, Ontario, Canada) were used with corresponding secondary antibodies (Dako, Ely, UK).
3
Western Blot Analysis of Autophagy Markers
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Antibodies were obtained from commercial sources: anti-NMDAR1, anti-NMDAR2B, and anti-LC3A/B; anti-APG5 and anti-ULK1 (Abcam, Cambridge, UK); anti-beclin1, anti-AMP-activated protein kinase (AMPK) α, and β-actin (Santa Cruz Biotechnology Inc., Dallas, TX, USA).
For the western blot analysis, the cells were rinsed with phosphate-buffered saline (PBS) and subsequently lysed for 30 min on ice in RIPA-B buffer (0.5% Nonidet P-40, 20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 50 mM NaF, 100 μM Na3VO4, 1 mM DTT, and 50 μg/mL PMSF). The insoluble material was removed by centrifugation at 12000 rpm for 20 min. Next, the supernatant was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blots were blocked in PBS with 5% skim milk and 0.05% Tween 20, incubated with the diluted primary antibodies against NMDAR1, NMDAR2B, or LC3A/B (1: 1000; Cell Signaling Technology, Denvers, MA, USA); APG5 or ULK1 (1: 1000; Abcam, Cambridge, UK); beclin-1 or AMPKα (1: 200; Santa Cruz Biotechnology Inc., Dallas, TX, USA), and followed by incubation with a horseradish peroxidase-conjugated secondary antibodies (1: 2500; Santa Cruz). β-actin (Santa Cruz) was used as a control. The blots were assayed using an enhanced chemiluminescence detection system (Image Quant LAS 4000mini, Pittsburgh, PA, USA).
For the western blot analysis, the cells were rinsed with phosphate-buffered saline (PBS) and subsequently lysed for 30 min on ice in RIPA-B buffer (0.5% Nonidet P-40, 20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 50 mM NaF, 100 μM Na3VO4, 1 mM DTT, and 50 μg/mL PMSF). The insoluble material was removed by centrifugation at 12000 rpm for 20 min. Next, the supernatant was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blots were blocked in PBS with 5% skim milk and 0.05% Tween 20, incubated with the diluted primary antibodies against NMDAR1, NMDAR2B, or LC3A/B (1: 1000; Cell Signaling Technology, Denvers, MA, USA); APG5 or ULK1 (1: 1000; Abcam, Cambridge, UK); beclin-1 or AMPKα (1: 200; Santa Cruz Biotechnology Inc., Dallas, TX, USA), and followed by incubation with a horseradish peroxidase-conjugated secondary antibodies (1: 2500; Santa Cruz). β-actin (Santa Cruz) was used as a control. The blots were assayed using an enhanced chemiluminescence detection system (Image Quant LAS 4000mini, Pittsburgh, PA, USA).
4
Immunoblotting and Immunofluorescence Protocols
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Commercially available antibodies were used for all immunoblotting and immunofluorescence studies. Anti‐ULK1, anti‐p‐ULK1 (Ser757), anti‐p‐ULK1 (Ser555), anti‐p‐mTOR, anti‐P62 and anti‐LC3 were obtained from Abcam (UK). Anti‐GAPDH was obtained from Shanghai Kangchen Bio‐tech Company (China). All secondary antibodies were obtained from Boster (China).
5
Western Blot Analysis of Autophagy Markers
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The cell samples were lysed by Radio-Immunoprecipitation Assay (RIPA) Buffer (Beyotime, China) containing phosphatase inhibitor cocktail and phenylmethylsulfonyl fluoride (PMSF) for protein extraction. The total cell proteins were denatured with SDS-PAGE loading buffer, separated by SDS-PAGE gel and transferred to Polyvinylidene Fluoride (PVDF) membrane (Millipore, USA). Then the membranes were blocked with 10% BSA and incubated with primary antibodies including anti-ULK1 (Abcam, USA), anti-Beclin-1 (Abcam, USA), anti-LC3 (Abcam, USA), p-Erk1/2 (CST, USA), Erk1/2 (CST, USA) and anti-β-actin (CST, USA) overnight at 4 °C. Subsequently, the blots were washed with TBST (Tris Buffered Saline with 0.05% Tween 20) three times and incubated with the Horseradish Peroxidase (HRP) conjugated secondary antibodies (1:10000, CST) for 45 min. The chemiluminescence reagent (Thermo, USA) was used to expose and identify the protein bands using ChemiDoc Touch System (Bio-Rad, USA), and quantitative analysis of the protein expression level was calculated relative to the β-actin control using the ImageJ software (NIH, USA).
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