For artificial cerebrospinal fluid (aCSF) preparation, NaCl (S7653) KCl (P9333), CaCl2(C5080), NaH2PO4(S8282), NaHCO3(S5761), sucrose (S7903), and ascorbate (A5960) were purchased from Sigma- Aldrich (St. Louis, MO). The concentration of the ingredients in the freshly prepared aCSF were: (in mM) 124 NaCl, 3 KCl, 1.5 MgSO47H20, 1.2 NaH2PO4, 2.4 CaCl2, 25 NaHCO3, and 10 D-Glucose. Neuronal Protein Extraction Reagent (N-PER) (Cat# 87792) was purchased from Thermo Fisher Scientific Inc. (Rockford, IL). 4X Laemmli buffer and Tris-buffered saline were purchased from Bio-Rad (Hercules, CA). Antibodies were purchased from Cell Signaling Technology (Danvers, MA). Ultrapure water was obtained from a Millipore Milli-Q synthesis system. (Billerica, MA). MgSO4 (AC M65500) and glucose (97061-164) were purchased from Acros organics and VWR, respectively.
N per neuronal protein extraction reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
The N-PER Neuronal Protein Extraction Reagent is a laboratory product designed for the extraction of proteins from neuronal cells. It is a ready-to-use solution that facilitates the isolation of neuronal proteins for further analysis and study.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
Mouse rat porcine canine tgf beta 1 quantikine elisa kit, by R&D Systems (3 mentions)
Fluostar omega, by BMG Labtech (3 mentions)
Mouse il 10 quantikine elisa kit, by R&D Systems (3 mentions)
Clarity western ecl substrate, by Bio-Rad (3 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (3 mentions)
Laemmli buffer, by Bio-Rad (2 mentions)
Tris buffered saline (tbs), by Bio-Rad (2 mentions)
Halt phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Pierce protease and phosphatase inhibitor mini tablet, by Thermo Fisher Scientific (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Synergy mx, by Agilent Technologies (2 mentions)
Emax immunoassay system, by Promega (2 mentions)
Aprotinin, by Santa Cruz Biotechnology (2 mentions)
Leupeptin, by Santa Cruz Biotechnology (2 mentions)
Ecl prime western blotting system, by Merck Group (2 mentions)
Non fat dry milk, by Bio-Rad (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Abcam (2 mentions)
Chemidoc system, by Bio-Rad (2 mentions)
Phosstop, by Merck Group (2 mentions)
35 protocols using n per neuronal protein extraction reagent
1
Artificial Cerebrospinal Fluid Preparation
2
Western Blotting Cerebellum Protein Analysis
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Western blotting was performed as described [18 (link), 27 (link)–29 ]. Briefly, cerebella were homogenized in ice-cold Neuronal Protein Extraction Reagent (N-PER; Thermo Fisher Scientific), containing Halt Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific), following the manufacturer’s protocol. Protein lysates (20–50 μg) were resolved on NuPAGE 4–12% Bis-Tris protein gels and transferred to Immobilon-P transfer polyvinylidene difluoride (PVDF) membranes (0.45 μm; Merck) for 1 h at 100 V (fixed) at 10°C, using a TE22 Hoefer transfer system (Hoefer Inc., Holliston. MA, USA). To monitor the levels of transferred proteins, blots were stained with Ponceau S (Merk). PVDF membranes were then incubated with the primary and secondary antibodies (see Supplementary Table 1) and analyzed using a ChemiDoc XRS_Plus instrument (Bio-Rad, Hercules, CA, USA).
3
Measuring Cytokine Levels in Neonatal Hypoxic-Ischemic Brain
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Ipsilateral hemispheres were collected from the pups at P13 after administering the HI insult at P10. Tissue samples were homogenized in N-PER™ Neuronal Protein Extraction Reagent (Thermo Fisher) with cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich) using pellet pestles, blue polypropylene (Sigma-Aldrich) on ice. After 1-h agitation at 4 °C, lysates were centrifuged at 14000 rpm for 10 min. The supernatant’s protein concentration was measured using the Pierce BCA Protein Assay kit (Thermo Scientific) according to the manufacturer’s protocol. Lysate aliquots were stored at − 80 °C for subsequent analysis. After the preparation of all the samples, the IL-10 and TGF-β levels in the lysates were measured using Mouse IL-10 Quantikine ELISA Kit (R&D system) and Mouse/Rat/Porcine/Canine TGF-beta 1 Quantikine ELISA Kit (R&D system). For TGF-β assessments, the samples were treated with 1N HCL to activate the latent TGF-β before measurement using an ELISA kit. The optical density was determined at 450 nm using a microplate reader (FLUOstar Omega, BMG LABTECH, Germany) within 15 min after stopping the reactions. All measurements were performed in duplicate.
4
Neuronal Protein Extraction and Analysis
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BBG, ethidium bromide, ATP, paraformaldehyde (PFA), RNAlater and glycerol gelatin were from Sigma-Aldrich (St. Louis, MO, USA). Sterile 0.9% NaCl was from Fresenius Kabi (Bad Homburg, Germany). Agarose was from Bioline (Alexandria, Australia). Ammonium-chloride-potassium (ACK) lysing buffer, N-PER™ neuronal protein extraction reagent, 100 × Halt™ protease inhibitor single-use cocktail, normal horse serum (NHS), DNase/RNase-free distilled water and SuperSignal West Pico Chemiluminescent Substrate were from ThermoFisher Scientific (Waltham, MA, USA). Foetal bovine serum (FBS) was from Lonza (Basel, Switzerland). Tissue-Tek® optimal cutting temperature (OCT) compound was from Sakura (Flemingweg, Netherlands) and 22 mm glass coverslips were from Menzel Glaser (Braunschweig, Germany). Diploma full-cream milk powder was from Fonterra (Mount Waverley, Australia). Bovine serum albumin (BSA) and all other reagent grade chemicals and salts were from Amresco (Solon, OH, USA). The antibodies used are listed in Table 1 .
5
Western Blot Analysis of Brain Samples
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Brain sample for Western blot assessment were obtained 1 day after reperfusion. Mice were transcardially perfused with ice-cold PBS and ipsilateral brain hemispheres were homogenized using N-PER neuronal protein extraction reagent (Thermo Scientific, IL, USA). Brain lysates containing 20 μg of total protein was separated by 10% SDS-PAGE and electrically transferred to nitrocellulose membranes. The membranes were blocked with 0.1% polyvinyl alcohol in Tris-buffered saline (TBS, pH 7.6) containing 0.2% Tween 20 for 30 min. The membranes were incubated with primary antibodies against rabbit p-IKKα/β (1:1000), rabbit p-IκBα (1:1000), rabbit IκBα (1:1000), rabbit 4-HNE (1:1000), mouse α-tubulin (1:5000), and mouse β-actin (1:5000), respectively, overnight at 4°C, followed by the further incubation with appropriate peroxidase-conjugated secondary antibodies (Invitrogen) for 2 h at room temperature. Specific bands were detected using the ECL western blotting substrate (Thermo Scientific) and exposed to LAS-4000 image detection system (Fuji, Japan).
6
Synaptosomal and Cytosolic Fractionation from Brain
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To isolate synaptosomal and cytosolic fractions from brain regions of interest, the Syn-PER Synaptic Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA, USA) was used. The isolated synaptosomes were lysed in the N-PER Neuronal Protein Extraction Reagent (Thermo Fisher Scientific) prior to any downstream applications, and all extraction reagents were supplemented with Pierce Protease and Phosphatase Inhibitor Mini Tablets (Thermo Fisher Scientific), as previously described [14 (link)]. We previously validated the efficacy of the synaptosomal/cytosolic extraction method by probing for proteins that are enriched in the corresponding fractions [14 (link)].
7
VEGF Measurement in Brain and Muscle
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Four days and 14 days after EMS surgery, brains and temporal muscles were quickly harvested after decapitation with overdosed pentobarbital (150 mg/kg, intraperitoneal). Thirty milligrams each of brain cortex and muscles tissue was obtained. Brain tissue was homogenized using N-PER Tissue Protein Extraction Reagent (Thermo Fisher Scientific Inc., Massachusetts, USA), and muscles were in N-PER Neuronal Protein Extraction Reagent (Thermo Fisher Scientific Inc., Massachusetts, USA). They were centrifuged at 10,000×g for 10 min at 4 °C, and the supernatant was obtained. One hundred microliters of the samples were applied to dish and VEGF protein level in each tissue (control group n = 7, HMGB1-treated group n = 7 at 4 days; control group n = 9, HMGB1-treated group n = 8 at 14 days) was measured using a rat VEGF ELISA assay kit (#27101 Rat VEFG Assay Kit, Immuno-Biological Laboratories Co., Ltd., Gunma, Japan) according to the manufacturer’s instructions.
8
Quantitative Western Blot Analysis of Cultured Neuronal Cells
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Cultured neuronal cells prepared as described before were first washed with ice-cold PBS and lysed in N-PER Neuronal Protein Extraction Reagent (Thermo Fisher Scientific) containing protease and phosphatase inhibitors (Halt Phosphatase Inhibitor Cocktail and Halt Protease Inhibitor Cocktail; Thermo Fisher Scientific), for 20 min on ice. The lysates were clarified by centrifugation for 15 min at 4°C. Quantitation of the protein content in lysates was performed with Micro BCA Protein Assay Kit (Thermo Fisher Scientific) and spectrophotometry on an Epoch BioTek spectrophotometer. Samples containing 20 μg of protein were incubated with Laemmli sample buffer containing β-ME (Bio-Rad; Hercules, CA, USA) for 5 min at 95°C. Subsequently, the samples and protein markers were electrophoresed on a 10% polyacrylamide Bis-Tris Plus gel with MES running buffer and transferred onto a PVDF membrane. The membrane was blocked with 5% BSA in TBST and incubated overnight with primary mAb Aβ (NAB228; Thermo Fisher Scientific). After several washes in 0.1% Tris-buffered saline (TBS) Tween 20, blots were incubated with HRP-conjugated secondary antibodies for 1 h at RT and developed using enhanced chemiluminescence (Clarity Western ECL Substrate; Bio-Rad). The protein bands were visualized by ChemiDoc™ MP Imaging System (Bio-Rad).
9
Cre-dependent SRGAP2C-HA Protein Expression
10
Quantifying Hippocampal BDNF and GDNF in Rats
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Rats receiving unilateral intrahippocampal transplantation of hNSC‐derived EVs or sham surgery were euthanized at 4 weeks after surgery using isoflurane anesthesia. Brains were immediately extracted from the skull (N = 6‐8 per group) and the hippocampus was dissected from each cerebral hemisphere. Each hippocampus was weighed and transferred into 300 μL ice‐cold lysis buffer (N‐PER Neuronal Protein Extraction Reagent, Thermo Scientific Product number 23225) containing sodium orthovanadate (0.5 mM), pheyl‐methylsulfonyl fluoride (PMSF, 1 mM), aprotinin (10 μg/mL), and leupeptin (1 μg/mL; Santa Cruz Biotechnology, Santa Cruz, California, http://www.scbt.com ). Tissues were sonicated individually, centrifuged at 4°C and the supernatants were collected and diluted 1:5 with Dulbecco's phosphate‐buffered saline. The supernatants were acidified to pH 2.6 then neutralized to pH 7.6, and the BDNF and GDNF levels were assayed using Emax ImmunoAssay Systems from Promega (BDNF catalog number G7611, GDNF catalog number G7621) and uncoated ELISA plates (Biolegend Nunc MaxiSorp, catalog number 423501). All measurements were performed at a wavelength of 450 μm on a microplate reader (BioTek Synergy Mx).
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