Autophagy Promotes Peripheral Nerve Regeneration and Motor Recovery Following Sciatic Nerve Crush Injury in Rats methods
Show snapshot details
On this page
This experiment, in seven questions
Jump straight to the part of the recipe you need. Data and provenance labels stay close to the action they support.
Shopping and prep list
What do I need before I start?
Biological model pending
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Before you run
What should be confirmed before execution?
First confirmation
Species or subject information is missing.
Confirm before execution
Protocol does not list structured equipment.
Confirm before execution
This page shows passages quoted from the paper. Equipment, materials, times, and temperatures are hidden until the page is rebuilt from the full paper and checked by a reviewer. Check the paper before using this method.
Confirm before execution
Open the source paper before finalizing run-specific details.
Procurement checkpoint
Use source-stated vendors where present. Treat mapped products as sourcing options unless the page marks an exact source match.
Open quote workflowStep-by-step procedure
What do I do, in order?
Drug Treatment
Animals were divided into four groups: sham + vehicle (v), crush + v, crush + rapamycin (rapa), and crush +3-methyladenine (3-MA). Rapamycin and 3-MA were obtained from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in 0.5 % dimethyl sulfoxide (DMSO). Animals received daily intraperitoneal injections of rapamycin at a dose of 1 mg/kg (crush + rapa group), 3-MA at a dose of 50 mg/kg (crush +3-MA group), or DMSO (crush + v group) for 5 days after the surgery. Sham-operated rats (sham + v group), in which the sciatic nerve was exposed but not subjected to crush injury, received daily intraperitoneal injections of an equal volume (1 mL) 0.5 % DMSO for 5 days. Drug dosages were selected based on data from previous studies and preliminary experiments (Maeda et al.; Chen et al.; Sekiguchi et al. ).
Immunocytochemistry
Transverse frozen sections (10-µm thickness) of sciatic nerve were dried, mounted onto slides, and washed in 0.1 M PBS and then blocked in 10 % goat serum and 3 % bovine serum albumin (BSA) for 1 h at 37 °C. After two washes with PBS, the slides were incubated with primary antibodies against MBP (1:1000; Sigma), NF-200 (1:500; Sigma), or LC3B-II (1: 500; Cell Signaling Technology, Danvers, MA, USA) at 4 °C for 12 h, followed by repeated washes with PBS, and then incubated with Alexa 488- or Cy3-conjugated secondary antibodies (1:1000; Invitrogen, Shanghai, China) for 2 h at room temperature. Sections were visualized under an epifluorescence microscope (Leica, DM6000B, Wetzlar, Germany) and imaged at × 400 magnification.
Western blot Analysis
LC3-II protein expression at the lesion site following sciatic NCI was analyzed by Western blotting. Nerve tissue samples were homogenized in tissue lysis buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1 % Triton X-100; 25 mM NaPPi; 80 mM β-glycerophosphate; 2 mM EDTA; 0.2 mM Na3VO4 and protease inhibitor cocktail) and centrifuged at 12,000 g for 15 min at 4 °C. The protein concentration in the supernatant was measured with a bicinchoninic assay kit (Beyotime Institute of Biotechnology, Shanghai, China), and the supernatant was mixed with sample buffer (50 mM Tris-HCl, 10 % sodium dodecyl sulfate, 10 % glycerol, 10 % 2-mercaptoethanol, and 2 mg/ml bromophenol blue) at a 1:1 ratio and boiled for 5 min. A total of 20 µg of each sample was separated on a 15 % acrylamide gel and transfer...
Measurement outputs
What raw and processed outputs should exist?
Protein expression by western blot
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Microscopy-based tissue or cell-state imaging
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Review raw outputs for quality, remove unusable captures, and organize the data into a comparison-ready table or image set.
inferred from protocolScoring or quantification
Quantify the primary readouts for this experiment: Protein expression by western blot; Microscopy-based tissue or cell-state imaging.
from paperStatistical comparison
Statistical method not yet structured for this page.
inferred from protocolReporting output
Report representative outputs alongside summary comparisons for Protein expression by western blot, Microscopy-based tissue or cell-state imaging.
inferred from protocolSource and audit
What supports the facts on this page?
Evidence quotes (3)
Animals were divided into four groups: sham + vehicle (v), crush + v, crush + rapamycin (rapa), and crush +3-methyladenine (3-MA). Rapamycin and 3-MA were obtained from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in 0.5 % dimethyl sulfoxide (DMSO). Animals received daily intraperitoneal injections of rapamycin at a dose of 1 mg/kg (crush + rapa group), 3-MA at a dose of 50 mg/kg (crush +3-MA group), or DMSO (crush + v group) for 5 days after the surgery. Sham-operated rats (sham + v group), in which the sciatic nerve was exposed but not subjected to crush injury, received daily intraperitoneal injections of an equal volume (1 mL) 0.5 % DMSO for 5 days. Drug dosages were selected based on data from previous studies and preliminary experiments (Maeda et al.; Chen et al.; Sekiguchi et al. ).
Transverse frozen sections (10-µm thickness) of sciatic nerve were dried, mounted onto slides, and washed in 0.1 M PBS and then blocked in 10 % goat serum and 3 % bovine serum albumin (BSA) for 1 h at 37 °C. After two washes with PBS, the slides were incubated with primary antibodies against MBP (1:1000; Sigma), NF-200 (1:500; Sigma), or LC3B-II (1: 500; Cell Signaling Technology, Danvers, MA, USA) at 4 °C for 12 h, followed by repeated washes with PBS, and then incubated with Alexa 488- or Cy3-conjugated secondary antibodies (1:1000; Invitrogen, Shanghai, China) for 2 h at room temperature. Sections were visualized under an epifluorescence microscope (Leica, DM6000B, Wetzlar, Germany) and imaged at × 400 magnification.
LC3-II protein expression at the lesion site following sciatic NCI was analyzed by Western blotting. Nerve tissue samples were homogenized in tissue lysis buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1 % Triton X-100; 25 mM NaPPi; 80 mM β-glycerophosphate; 2 mM EDTA; 0.2 mM Na3VO4 and protease inhibitor cocktail) and centrifuged at 12,000 g for 15 min at 4 °C. The protein concentration in the supernatant was measured with a bicinchoninic assay kit (Beyotime Institute of Biotechnology, Shanghai, China), and the supernatant was mixed with sample buffer (50 mM Tris-HCl, 10 % sodium dodecyl sulfate, 10 % glycerol, 10 % 2-mercaptoethanol, and 2 mg/ml bromophenol blue) at a 1:1 ratio and boiled for 5 min. A total of 20 µg of each sample was separated on a 15 % acrylamide gel and transferred to a nitrocellulose membrane (BioScience, Shanghai, China) by electroblotting (Bio-Rad, Hercules, California, USA) at 120 V for 1.5 h followed by 70 V for 0.5 h at 4 °C. Membranes were blocked with 3 % BSA and incubated with rabbit anti-LC3B-II antibody (1:000)...
Machine-readable layer
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Autophagy Promotes Peripheral Nerve Regeneration and Motor Recovery Following Sciatic Nerve Crush Injury in Rats methods",
"description": "Protocol for Autophagy Promotes Peripheral Nerve Regeneration and Motor Recovery Following Sciatic Nerve Crush Injury in Rats methods",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Drug Treatment",
"text": "Animals were divided into four groups: sham + vehicle (v), crush + v, crush + rapamycin (rapa), and crush +3-methyladenine (3-MA). Rapamycin and 3-MA were obtained from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in 0.5 % dimethyl sulfoxide (DMSO). Animals received daily intraperitoneal injections of rapamycin at a dose of 1 mg/kg (crush + rapa group), 3-MA at a dose of 50 mg/kg (crush +3-MA group), or DMSO (crush + v group) for 5 days after the surgery. Sham-operated rats (sham + v group), in which the sciatic nerve was exposed but not subjected to crush injury, received daily intraperitoneal injections of an equal volume (1 mL) 0.5 % DMSO for 5 days. Drug dosages were selected based on data from previous studies and preliminary experiments (Maeda et al.; Chen et al.; Sekiguchi et al. )."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Immunocytochemistry",
"text": "Transverse frozen sections (10-µm thickness) of sciatic nerve were dried, mounted onto slides, and washed in 0.1 M PBS and then blocked in 10 % goat serum and 3 % bovine serum albumin (BSA) for 1 h at 37 °C. After two washes with PBS, the slides were incubated with primary antibodies against MBP (1:1000; Sigma), NF-200 (1:500; Sigma), or LC3B-II (1: 500; Cell Signaling Technology, Danvers, MA, USA) at 4 °C for 12 h, followed by repeated washes with PBS, and then incubated with Alexa 488- or Cy3-conjugated secondary antibodies (1:1000; Invitrogen, Shanghai, China) for 2 h at room temperature. Sections were visualized under an epifluorescence microscope (Leica, DM6000B, Wetzlar, Germany) and imaged at × 400 magnification."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Western blot Analysis",
"text": "LC3-II protein expression at the lesion site following sciatic NCI was analyzed by Western blotting. Nerve tissue samples were homogenized in tissue lysis buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1 % Triton X-100; 25 mM NaPPi; 80 mM β-glycerophosphate; 2 mM EDTA; 0.2 mM Na3VO4 and protease inhibitor cocktail) and centrifuged at 12,000 g for 15 min at 4 °C. The protein concentration in the supernatant was measured with a bicinchoninic assay kit (Beyotime Institute of Biotechnology, Shanghai, China), and the supernatant was mixed with sample buffer (50 mM Tris-HCl, 10 % sodium dodecyl sulfate, 10 % glycerol, 10 % 2-mercaptoethanol, and 2 mg/ml bromophenol blue) at a 1:1 ratio and boiled for 5 min. A total of 20 µg of each sample was separated on a 15 % acrylamide gel and transfer..."
}
],
"tool": [],
"supply": [],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "Autophagy Promotes Peripheral Nerve Regeneration and Motor Recovery Following Sciatic Nerve Crush Injury in Rats",
"datePublished": "2016",
"author": [
{
"@type": "Person",
"name": "Hai-cheng Huang"
},
{
"@type": "Person",
"name": "Li Chen"
},
{
"@type": "Person",
"name": "Hai-xing Zhang"
},
{
"@type": "Person",
"name": "Sheng-fa Li"
},
{
"@type": "Person",
"name": "Pei Liu"
},
{
"@type": "Person",
"name": "Tian-yun Zhao"
},
{
"@type": "Person",
"name": "Chuan-xiang Li"
}
],
"identifier": "10.1007/s12031-015-0672-9"
}
},
{
"@context": "https://schema.org",
"@type": "BreadcrumbList",
"itemListElement": [
{
"@type": "ListItem",
"position": 1,
"name": "Experiments",
"item": "https://replicatescience.com/experiments"
},
{
"@type": "ListItem",
"position": 2,
"name": "Autophagy Promotes Peripheral Nerve Regeneration and Motor Recovery Following Sciatic Nerve Crush Injury in Rats methods",
"item": "https://replicatescience.com/experiments/autophagy-promotes-peripheral-nerve-regeneration-and-motor-recovery-following-sciatic-nerve-crush-injury-in-rats-methods-hai-cheng-huang-pmc4829621/autophagy-promotes-peripheral-nerve-regeneration-and-motor-recovery-following-sciatic-nerve-crush-in-mlph9cpn"
}
]
}
]