Under deep anesthesia with sodium pentobarbital at a dose of 60 mg kg−1, six male SD rats from each experimental group were transcardially perfused with 500 ml NS, followed by 500 ml 4% paraformaldehyde in PBS. The spinal cord was removed and post-fixed in 10% formalin solution for 12 h at 4°C. All samples were embedded in paraffin and transversely cut into 4-µm thick sections with a sliding microtome. Following deparaffinization and hydration, sections were blocked in 3% H2O2 for 10 min at room temperature, and were incubated with primary antibodies, including anti-NR2B (ab216621; 1:250; Abcam, Cambridge, UK), anti-p-CaMKIIα (ab5683; 1:300; Abcam), and anti-p-CREB (9198; 1:700; Cell Signaling Technology, Inc.), for 2 h at 37°C. Slides were washed with 0.1 M PBS 3 times for 2 min and subsequently incubated with a goat anti-rabbit secondary antibody (PV-9001; 1:500; ZSGB-BIO; OriGene Technologies, Inc., Rockville, MD, USA) for 30 min at 37°C, then stained with diaminobenzidine (DAB kit; ZSGB-BIO; OriGene Technologies, Inc.) and counterstained with hematoxylin for 30 sec at room temperature. Images were captured with a light microscope (Olympus Corporation, Tokyo, Japan). Quantitative image analysis of the relative optical density (OD) was performed using Image Pro Plus software version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).
Ab5683
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab5683 is a primary antibody product from Abcam. It is a polyclonal antibody targeting a specific protein of interest. The antibody is produced in a host species and purified for use in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab52476, by Abcam (3 mentions)
Ab18258, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Pv 9001, by OriGene (1 mentions)
Anti pcreb, by Cell Signaling Technology (1 mentions)
Image pro plus software version 6, by Media Cybernetics (1 mentions)
Ab137869, by Abcam (1 mentions)
Ab8805, by Abcam (1 mentions)
Kn 62, by Merck Group (1 mentions)
Mab1018, by R&D Systems (1 mentions)
Ab81283, by Abcam (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Ab9485, by Abcam (1 mentions)
Ab11959, by Abcam (1 mentions)
Alexa fluor 488 donkey anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
Cm1950, by Leica (1 mentions)
Alexa fluor 594 goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Ab51243, by Abcam (1 mentions)
Ab45689, by Abcam (1 mentions)
Ab109520, by Proteintech (1 mentions)
11 protocols using ab5683
1
Immunohistochemical Analysis of Spinal Cord Proteins
2
Chronic Low-Dose Radiation Effects on PC12 Cells
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We utilized the following reagents to investigate the effects of chronic, low-dose-rate 137Cs-γ radiation on NGF-induced neurite extension in PC12 cells: NGF 2.5S (N-100, Alomone Labs, Jerusalem, Israel); anti-ERK1/ERK2 mouse-monoclonal antibody (MAB1576) and anti-phosphorylated ERK1/ERK2 rabbit-polyclonal antibody (MAB1018, both R & D systems, Minneapolis, MN, USA); anti-Trk A rabbit polyclonal antibody (sc-118) and anti-phosphorylated Trk A mouse-monoclonal antibody (sc-8058, both Santa Cruz Biotechnology, Dallas, TX, USA)—Trk A is a high-affinity nerve growth factor receptor; anti-tyrosine hydroxylase rabbit-monoclonal antibody (ab137869); anti-Akt rabbit polyclonal antibody (ab8805) and anti-phosphorylated Akt rabbit monoclonal antibody (ab81283); anti-Ca2+/calmodulin-dependent kinase II (CaMKII) rabbit polyclonal antibody (ab131468) and anti-phosphorylated CaMKII rabbit polyclonal antibody (ab5683, all Abcam plc, Cambridge, UK); KN-62 (I2142, Sigma-Aldrich, St Louis, MO, USA).
3
Quantification of synaptic proteins in HT22 cells
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RIPA lysis buffer containing phenylmethylsulfonyl fluoride was added to lyse HT22 cells, and proteins were extracted for quantification. Proteins were separated using SDS-PAGE and transferred onto a PVDF membrane. Then, the membranes were blocked with 5% milk for 2 h and incubated overnight at 4°C with the following primary antibodies: anti-PSD95 (ab18258, Abcam, United States), anti-p-CaMKII (ab5683, Abcam, United States), anti-CaMKII (ab52476, Abcam, United States), and anti-GAPDH (ab9485, Abcam, United States). Subsequently, they were incubated with DylightTM 800 goat anti-rabbit fluorescent secondary antibodies (611–145-002, Rockland, United States) in the dark for 2 h. Finally, imaging analysis was performed using an Odyssey imaging system (LICOR, United States). The relative expression of the target protein was calculated using the grey value of GADPH as a reference.
4
Immunofluorescent Labeling of Brain Slices
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Take out the dehydrated brain, cut it perpendicularly to the missing seam, place it on the stage, and embed it in OCT. Using a cryostat (Leica CM1950), put it in a microtome and quickly freeze it at −20°C for 20 min. Then transfer it to the slicing table, perform coronal sectioning with a thickness of 30 um, gently pick it out with a brush, and place it in a six-well plate of 0.1 M PBS. Three sets of brain slices were selected for each group, and rinsed for three times at room temperature with 0.1MPBS (pH 7.4) for 10 min each time. Then place the brain slices in the antibody diluent (3% BSA, 0.3% Triton X-100 PBS) that has been added to the primary antibody, and incubate at room temperature for 16 h on a shaker. Use the following primary antibodies: mouse anti c-fos (1:500; abcam, ab11959), rabbit anti CaMKII (1:200; abcam, ab5683). The brain slices were rinsed three times with 0.1MPBS at room temperature for 10 min each time, and then the brain slices were placed in the antibody diluent with the added secondary antibody, incubated for 4 h, and DAPI was added at 3.5 h. Use the following secondary antibodies: Donkey Anti-Rabbit IgG H&L Alexa Fluor® 488 (1:500; invitrogen, A-21206), Goat Anti-Mouse IgG H&L Alexa Fluor® 594 (1:500; invitrogen, A-21202), DAPI (1:1000; Sigma, D9542). Then the brain slices were rinsed for three times with 0.1MPBS for 10 min each time.
5
Protein Expression Analysis of hAMSCs and BMSCs
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The total protein from hAMSCs and BMSCs was extracted with RIPA lysis buffer and quantified with BCA Protein Assay Kit (Beyotime). Proteins were separated using SDS‐PAGE and subsequently electrotransferred onto polyvinylidene fluoride membranes. The antibodies used were as follows: Anti‐p16 antibody (Abcam; ab51243), anti‐p21 antibody (Abcam; ab109520 and Proteintech; 28248‐1‐AP), anti‐Nrf2 antibody (Abcam; ab62352), anti‐HO‐1 antibody (Huabio), anti‐NQO1 antibody (Huabio), anti‐14‐3‐3ε antibody (Huabio), anti‐calmodulin (CaM) antibody (Abcam; ab45689), anti‐CaMKII antibody (Abcam; ab52476), anti‐CaMKII (phospho T286) antibody (Abcam; ab5683), and anti‐Lamin B (Abcam; ab133741). TBST was used to dilute these antibodies. Subsequently, the membranes were incubated with a horseradish peroxidase‐conjugated secondary antibody (Protein Tech; SA00001‐2) for 120 min at 24–28°C. Finally, the enhanced chemiluminescence hypersensitive luminescent solution (Beyotime) was used to treat the membranes, which were imaged using the Chemi DocTM MP Imaging System (Bio‐Rad). ImageJ software was used to analyze the intensity of the protein bands.
6
Western Blot Analysis of Signaling Proteins
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Western blot procedures were performed as previously described [16 ]. The primary antibodies were rabbit anti-calcium/calmodulin-dependent protein kinase II (CaMKII) (1 : 1000, ab52476, Abcam, Cambridge, United Kingdom), rabbit anti-phospho- (P-) CaMKII (1 : 1000, ab5683, Abcam), rabbit anti-AKT (1 : 1000, #4685, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-P-AKT (1 : 1000, #4060, Cell Signaling Technology), rabbit anti-P-ERK1/2 (1 : 1000, #4370, Cell Signaling Technology), rabbit anti-ERK1/2 (1 : 1000, #4695, Cell Signaling Technology), rabbit anti-P-p38 (1 : 1000, #9215, Cell Signaling Technology), rabbit anti-p38 (1 : 1000, #9212, Cell Signaling Technology), rabbit anti-P-SAPK/JNK (1 : 1000, #4668, Cell Signaling Technology), rabbit anti-SAPK/JNK (1 : 1000, #9258, Cell Signaling Technology), rabbit anti-TLR4 (1 : 1000, #14358, Cell Signaling Technology), rabbit anti-NF-κB p65 (1 : 1000, #8242, Cell Signaling Technology), rabbit anti-P-NF-κB p65 (1 : 1000, #3033, Cell Signaling Technology), and rabbit anti-β-actin (1 : 2000, #4970, Cell Signaling Technology). Horseradish peroxidase- (HRP-) conjugated goat anti-rabbit IgG (1 : 5000, A8275, Sigma-Aldrich) was used as a secondary antibody.
7
Immunofluorescence Analysis of PSD95 and p-CaMKII
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HT22 cells plated on coverslips were fixed with 4% paraformaldehyde at room temperature for 15 min and sealed with 10% donkey serum at room temperature for 1 h. Afterward, the cells were incubated overnight at 4°C with the following primary antibodies: anti-PSD95 (ab18258, Abcam, United States) or anti-p-CaMKII (ab5683, Abcam, United States), incubated with donkey anti-rabbit fluorescent secondary antibody (A21206, Invitrogen, United States) for 2 h at room temperature in the dark, and counterstained with DAPI (Sigma, United States) for 10 min. Fluorescence images were obtained by using an inverted Olympus FV1200 confocal microscope system. The relative mean fluorescence intensity was measured using Image-Pro Plus software (Media Cybernetics).
8
Immunohistochemical Labeling of Brain Regions
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All animals were anesthetized (Avertin, 13 ml/g, intraperitoneally) and perfused intracardially with 0.9% saline followed by 4% paraformaldehyde in phosphate-buffered saline (PBS). Brains were removed. For CaMKIIα labeling, 40 μm cryostat sections containing the LHb were placed in blocking solution for 1 h before incubation in primary antibody against CaMKIIα (rabbit, 1:500; ab5683, Abcam) (36 h at 4°C). Sections were then incubated with corresponding secondary antibody at a dilution of 1:400 for 6 h at room temperature: (Dylight 594) goat-anti-rabbit IgG (DI-1649, Vector Laboratories). For GABA labeling, 40 μm cryostat sections containing the RMTg were placed in blocking solution for 1 h before incubation in primary antibody against GABA (rabbit, 1:500; A2052, sigma) (36 h at 4°C). Sections were then incubated with corresponding secondary antibody at a dilution of 1:400 for 6 h at room temperature: (Dylight 594) goat-anti-rabbit IgG (DI-1649, Vector Laboratories).
9
Western Blot Analysis of Kinase Signaling
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Sixty seconds after hypotonic or hypertonic medium treatment in the presence or absence of kinase inhibitors, HUVEC cells grown on 6‐cm dishes were washed with ice‐cold phosphate‐buffered saline and resuspended with cell lysis buffer (Beyotime, Shanghai, China) including Halt™ Phosphatase Inhibitor Cocktail (Thermo Fisher, Shanghai, China) and homogenized by 5 passages through a 27‐gauge needle. The homogenate was centrifuged at 13 000 g for 20 min at 4 °C. The supernatant was collected, and protein concentration was measured using the Bradford protein assay method (Beyotime). Ten micrograms of total protein was resolved on a 10% SDS‐polyacrylamide gel and transferred onto a polyvinylidene fluoride membrane. The membranes were blocked with 5% nonfat milk in PBST for 30 min at room temperature and then incubated with antibodies against phospho‐PKC (ab23513, Abcam, Cambridge, MA), PKC (ab221611, Abcam), phospho‐CaMKII (ab5683, Abcam), CaMKII (ab22609, Abcam) overnight at 4 °C. After washing, the membranes were incubated with anti‐rabbit IgG horseradish peroxidase secondary antibody (Amersham Biosciences, Piscataway, NJ, USA). The immunoreactive bands were visualized using enhanced chemiluminescence method (PerkinElmer Life Sciences, Waltham, MA, USA).
10
CaMKIIα Phosphorylation Quantification
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L4-L6 DRGs were harvested, lysed in RIPA buffer and cleared by centrifugation at 12,000 rpm for 20 min. After quantification, the protein samples (25 ug) were resolved by 8% SDS-PAGE before transfer onto PVDF membranes. Membranes were blocked with 5% milk-TBST for 1 h at 37 ℃ and then incubated with rabbit anti-p-CaMKIIα (ab5683, 1:1000, Abcam, USA), mouse anti-CaMKIIα (50049, 1:1000, CST, USA), and mouse anti-β-actin (12262,1:5000, CST, USA) at 4 °C overnight. Membranes were washed with TBST for 5–10 min and then incubated with HRP-conjugated anti-rabbit (7074, 1:5000, CST, USA) or anti-mouse (7076,1:5000, CST, USA) IgG for 2 h at 37 °C. Signal was then developed by enhanced chemiluminescence (Beyotime, Shanghai, China) following manufacturer instructions. The protein bands were analyzed using ImageJ software and normalized to β-actin.
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