The total protein of the spinal cord tissues was extracted using RIPA lysis buffer. Total proteins of each group (40 μg) were resolved in 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidenefluoride (PVDF) membranes (Millipore, Bedford, MA). After the membranes were blocked in5% non-fat milk at room temperature for 30 min, they were incubated with anti-AQP4 (ab46182, Abcam, Cambridge, MA), anti-NKCC1 (ab59791, Abcam), or anti-β-actin (A2066, Sigma, St Louis, MO) antibody at 4°C overnight. After 3 washes with PBST, the membranes were incubated with proper horseradish peroxidase-conjugated goat anti-rabbit IgG antibody at room temperature for 60 min, and visualized with the enhanced chemiluminescence (ECL) substrate (ThermoFisher, Shanghai, China). The images were scanned and analyzed with ImageJ (NIH, Bethesda, MD).
Ab46182
Manufactured by Abcam
Sourced in United States
Ab46182 is a lab equipment product. It is a tool used for scientific research and experiments in a laboratory setting. The core function of this product is to perform a specific task or technique required in the research process.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Ab59791, by Abcam (2 mentions)
Ab8227, by Abcam (2 mentions)
Ab48004, by Abcam (2 mentions)
Ecl substrate, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg antibody, by Thermo Fisher Scientific (1 mentions)
A2066, by Merck Group (1 mentions)
Ix71 microscope, by Olympus (1 mentions)
Ab32575, by Abcam (1 mentions)
Ab168387, by Abcam (1 mentions)
Ab92547, by Abcam (1 mentions)
Mab3402, by Merck Group (1 mentions)
Enhanced chemiluminescence detection system, by Cell Signaling Technology (1 mentions)
Rapid gold bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Ab9512, by Abcam (1 mentions)
Ab2860, by Abcam (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
2 methylbutane, by Thermo Fisher Scientific (1 mentions)
Ab53554, by Abcam (1 mentions)
Alexa fluor 488 donkey anti rabbit igg antibody, by Thermo Fisher Scientific (1 mentions)
15 protocols using ab46182
1
Western Blot Analysis of AQP4 and NKCC1 in Spinal Cord
2
Spinal Cord Tissue Immunofluorescence Staining
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Paraffin-embedded spinal cord tissue at injury site was cut at a thickness of 6 micron. Slides were deparaffinized and rehydrated by washing in xylene and passing through ethanol gradient. Endogenous peroxidase activity was quenched with freshly made 0.3% hydrogen peroxide and antigens were heat retrieved in citrate buffer. Slides were blocked with normal goat serum at 37°C for 30 min, incubated with antibody against AQP4 (ab46182, Abcam) or NKCC1 (ab59791, Abcam) overnight at 4°C, rinsed with 0.01 M PBS 3 min for 3 times, incubated with FITC- (for AQP4) or Cy5- (for NKCC1) conjugated goat anti-rabbit IgG antibodies at 37°C for 30 min, conterstained with DAPI. The slides were dehydrated with alcohol gradient, xylene cleared, and mounted with neutral gum. The images were obtained with an Olympus IX71 Microscope.
3
Protein expression analysis by Western blot
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Western blot was used to detect the protein content of AQP1-4, kidney injury molecule-1 (KIM-1), α-smooth muscle actin (α-SMA), and vimentin. The steps were as previously described[26 (link)]. The primary antibodies used for Western blot were purchased from Abcam (KIM-1: ab190696; α-SMA: ab32575; vimentin: ab92547; AQP1: ab168387; AQP4: ab46182). Grey bands on the picture were semi-quantitatively analyzed with ImageJ, with GAPDH used as the internal control.
4
Quantitative Analysis of AQP4 and GFAP
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The perihematomal tissues were extracted and then homogenized in lysis buffer containing a protease inhibitor cocktail. The supernatant was collected after centrifugation at 12,000 rpm for 10 min. The protein concentration was determined using the rapid gold BCA protein assay kit (Thermoscientific, Rockford, United States). Equal amount of protein were separated through 10% SDS PAGE then transferred onto a PVDF membrane (Millipore, Billerica, MA, United States). The blots were incubated overnight at 4°C with anti-AQP4 (ab46182, abcam) and anti-GFAP (MAB3402, Millipore) antibodies. Protein bands were detected using an enhanced chemiluminescence detection system (Cell Signaling Technology, Beverly, MA, United States) and the intensities of immunoreactive bands were analyzed using an image analysis program (Image J, NIH, United States).
5
Quantification of AQP4 and Calmodulin Proteins
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AQP4 and calmodulin protein levels were measured by sandwich ELISA following the manufacturer's instructions (Abcam) and as described by Salman et al. (2017 ), using rabbit polyclonal anti‐AQP4 antibody (Abcam, ab46182) and mouse monoclonal anti‐AQP4 antibody (Abcam, ab9512) or rabbit polyclonal anti‐calmodulin antibody (Abcam, ab38590) with mouse monoclonal anti‐calmodulin antibody (Abcam, ab2860). The secondary antibody used in both assays was chicken anti‐mouse IgG‐HRP antibody (Santa Cruz, sc‐2954).
6
Fluorescent Immunohistochemistry of Brain Tissues
7
Quantifying Astrocytic AQP4 Protein Expression
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Western blot was employed to evaluate AQP4 protein expression in astrocytes. Total protein extract (50 μg) was loaded onto a 10% sodium dodecyl sulphate-polyacrylamide gel, separated by electrophoresis, and transferred onto a nitrocellulose membrane (Sartorius, Goettingen, Germany). After blocking with 5% milk for 1 hours, the membrane was incubated with polyclonal AQP4 (1:500, ab46182) or β-actin (1:1,000; ab8227) antibody (Abcam, Cambridge, UK) overnight at 4°C and then horseradish peroxidase-conjugated goat anti-rabbit polyclonal IgG (1:800; sc2004; Santa Cruz Biotechnology, Inc, Dallas, TX) for 1 hours at room temperature. The probed target proteins were then visualized with enhanced chemiluminescence reagent (Santa Cruz Biotechnology, Inc) and exposed on an X-ray film.
8
Intestinal Membrane Protein Analysis
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Cytomembrane protein and total protein were extracted from jejunum, ileum, and IPEC-J2 cells, using a membrane and cytosol protein extraction kit (Beyotime) and RIPA Lysis Buffer (Beyotime) in accordance with the manufacturer's instructions. Cytomembrane protein was used to determine the relative protein levels of AQP3 (sc-20811, Santa Cruz, TX, USA), AQP4 (ab46182, Abcam, LON, UK), AQP8 (sc-14984, Santa Cruz), epithelial sodium channel alpha subunit (α-ENaC, ab65710, Abcam), sodium-hydrogen exchanger 3 (NHE3, sc-28757, Santa Cruz), Cl−/HCO3− exchanger (DRA/PAT1, sc-161150, Santa Cruz), β-ENaC (14134-1-AP, Proteintech, CA, USA), and β-ATPase Na+/K+ antibody (ab254025, Abcam). Total protein was used to measure the relative protein levels of zonula occludens-1 (ZO-1, 21773-1-AP, Proteintech), occludin (ab31721, Abcam), E-cadherin (Proteintech), claudin-1 (#13995, Cell Signaling Technology, Boston, USA), claudin-2 (ab53032, Abcam), AMPKα1 (10929-2-AP, Proteintech), AMPKα2 (18167-1-AP, Proteintech), PGC-1α (ab106814, Abcam), target of rapamycin complex 2 (TORC2, 12497-1-AP, Proteintech), silent information regulator T1 (SIRT1,13161-1-AP, Proteintech), and β-actin (60008-1-Ig, Proteintech). These proteins were detected by the Western blotting technique as previously reported (Tan et al., 2010 (link)).
9
Astrocyte Protein Quantification Protocol
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After isolation from Matrigel, the astrocytes were lysed with lysis buffer (4 M urea, 5 mM EDTA, 0.5% SDS, 0.5% NP-40, 100 mM Tris pH 7.4) containing protease and phosphatase inhibitor (Sigma-Aldrich, St. Louis, MO). The homogenates were centrifuged at max speed for 5 minutes at 4 °C. Cell debris was withdrawn leaving just the supernatants followed by determination of protein concentration with a BCA protein assay. 40 μg/lane of protein was loaded onto a Criterion TGX Precast gel and subsequently transferred onto PVDF (polyvinyl difluoride) membrane. Following transfer, the membranes were blocked with Odyssey Blocking Buffer (LI-COR, Bioscence, Germany). The membranes were then probed with Rabbit anti-AQP4 antibody (Abcam, ab46182), anti-Connexin 43 (Abcam, ab11370) and Mouse anti-GAPDH (Abcam, ab8245) in Odyssey Blocking Buffer. GAPDH was used as the gel loading control. Both the AQP4 and GAPDH antibodies were used at a dilution of 1:500. Secondary antibodies (donkey anti-rabbit 800 and donkey anti-mouse 700) were used at a dilution of 1:20,000. After Western blotting, the protein bands were detected with a Li-cor Odyssey infrared scanner. Band integrated optical density was quantified using ImageJ software. Sample size was at least 5 for each treatment group.
10
Western Blot Analysis of Glial Markers
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