Tissue sections for subcellular localization of AQP4 in relation to cerebral blood vessels or astrocytes were processed as above and immunofluorescently stained for AQP4 (1:60, ab9512, Abcam), and either the blood vessel endothelial cell marker CD31 (1:25, ab28364, Abcam), or astrocyte marker GFAP (1:3000; PU020-UP, Biogenix). Following deparaffinization and antigen retrieval, tissue sections were incubated in a cocktail of primary antibodies overnight at 4°C [CD31, 1:25 (ab28364, Abcam); GFAP (1:3000; PU020-UP, Biogenix); AQP4, 1:60 (ab9512, Abcam)], washed in PBS-T and then incubated in a cocktail of fluorophore-conjugated goat anti-rabbit (Alexa Fluor® 488, 1:500; A11008) and goat anti-mouse (Alexa Fluor® 568, 1:500; A11004) secondary antibodies (Invitrogen) for 2 h at room temperature. Finally, sections were washed in PSB-T and then dH2O, before being cover-slipped in VECTASHIELD® mounting medium with DAPI (H-1200, Vector Labs). All incubations were performed manually and in a humidity tray, with minimal exposure to light. Immunofluorescence images were taken using a Leica DMLB fluorescent microscope and Q Capture Pro7 software (QImaging). Images were acquired by single excitation of each wavelength (separately) and channels subsequently merged.
Ab9512
Manufactured by Abcam
Sourced in United States
Ab9512 is a lab equipment product offered by Abcam. It is a device designed for laboratory use, but no further details about its core function can be provided in an unbiased and factual manner while maintaining conciseness.
Automatically generated - may contain errors
Lab products found in correlation
Ab16667, by Abcam (3 mentions)
Pa3030a, by Thermo Fisher Scientific (2 mentions)
Ab28346, by Abcam (2 mentions)
Ab84125, by Abcam (2 mentions)
Ab71204, by Abcam (2 mentions)
Ab58207, by Abcam (2 mentions)
Ab5690, by Abcam (2 mentions)
Ab183685, by Abcam (2 mentions)
Ab22378, by Abcam (2 mentions)
Ab6640, by Abcam (2 mentions)
Ab155101, by Abcam (2 mentions)
N4142, by Merck Group (2 mentions)
Sag4318, by Fujifilm (2 mentions)
Mbs767644, by MyBioSource (2 mentions)
Sc 18355, by Santa Cruz Biotechnology (2 mentions)
Ne1023, by Merck Group (2 mentions)
Af3628, by R&D Systems (2 mentions)
Ab53444, by Abcam (2 mentions)
Ab416, by Abcam (2 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
14 protocols using ab9512
1
Subcellular Localization of AQP4 in Brain
2
Immunohistochemical Profiling of Neural and Immune Markers
3
Immunohistochemical Profiling of Neural and Immune Markers
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The method has been described previously (29 (link)). Primary antibodies were to the following proteins: PDGFRα (1:200, rat, 558774, BD Biosciences), PDGFRα (1:8000, rabbit, gift from W. Stallcup, Sanford Burnham Prebys), Olig2 (1:2000, rabbit, gift from C.D. Stiles, Harvard), CD31 (1:200, rat, 553370, BD Biosciences), CD31 (1:200, rabbit, ab28346, Abcam), RNF43 (1:200, rabbit, ab84125, Abcam), WIF1 (1:200, rabbit, ab71204, Abcam), Caspase-3 (1:200, rabbit, 9661, Cell signaling), Aqp4 (1:100, mouse, ab9512, Abcam), PLVAP (1:100, rat, 553849, BD Pharmingen), Claudin5 (1:200, mouse, 352588, ThermoFisher), Fibrinogen (1:200, mouse, ab58207, Abcam), Iba1 (1:500, rabbit, SAG4318, Wako), CD11c (1:500, rabbit, MBS767644, MyBioSource), Arg1 (1:100, goat, sc-18355, Santa Cruz), iNOS (1:200, rabbit, PA3030A, ThermoFisher), CD3 (1:100, rabbit, ab5690, Abcam), CD4 (1:200, rabbit, ab183685, Abcam), CD8 (1:100, rat, ab22378, Abcam), F4/80 (1:500, rat, ab6640, Abcam), Ki67 (1:200, rabbit, ab16667, Abcam), SMI32 (1:1000, mouse, NE1023, Millipore), NF200 (1:1000, rabbit, N4142, Sigma), Wif1 (1:1000, rabbit, ab155101, Abcam).
4
Immunohistochemical Analysis of Brain Tissue
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The brains were removed after perfusion with the fixative 4% paraformaldehyde and then immersed in 30% sucrose in PBS. Serial sections were cut on a freezing microtome, blocked, and incubated with the following primary antibodies: goat anti‐CD31 (diluted 1:250, R&D, AF3628), rabbit anti‐Iba1 (diluted 1:500, Wako, 019‐19741), rat anti‐CD68 (diluted 1:100, Abcam, ab53444), rat anti‐C3 (diluted 1:100, Abcam, ab11862), mouse anti‐AQP4 (diluted 1:100, Abcam, ab9512), rabbit anti‐GFAP (diluted 1:500, Abcam, ab7260), and rabbit anti‐MMP9 (diluted 1:500, Abcam, ab38898). After washing, the sections were incubated with the appropriate Alexa Fluor 488‐ or Alexa Fluor 555‐conjugated secondary antibody (diluted 1:1000, Invitrogen) and counterstained with DAPI. Images were captured with a Zeiss microscope (Zeiss, LSM780).
5
Nephridium Protein Expression Analysis
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Total proteins from nephridium were extracted with RIPA lysis buffer, and equal amounts were then resolved using SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. After being blocked with 5% nonfat milk, the membranes were incubated overnight with primary antibodies, and the antibodies used included anti-GFAP (ab4648, Abcam), anti-AQP4 (ab9512, Abcam), anti-IL-1β (ab9722, Abcam), and anti-TNF-α (ab9755, Abcam). Next, the membranes were incubated with peroxidase-conjugated secondary antibodies. The optical densities of the bands were quantified using β-actin or GAPDH as the internal references, and immunocomplexes were visualized with an Odyssey near infrared dual color laser imaging system.
6
Dual Immunolabeling of AQP4 and ANGPT1
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Immunolabeling was performed using the same protocol as was described above for NOTCH1 (single labeling). Primary antibody used were mouse monoclonal antibody synthetic AQP4 (1:100, #ab9512, Abcam) and rabbit-polyclonal antibody raised against human ANGPT1 (1:200, ab102015, Abcam). Specificity of antibodies has been demonstrated previously [29 (link)–30 (link)].
7
Immunofluorescence Analysis of Mouse Brain Tissue
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For immunofluorescence of mouse tissue, tumor bearing mice were cardially perfused with PBS, followed by 4.5% phosphate-buffered formaldehyde solution (Roti-Histofix 4.5%, 2213, Carl Roth). Brain tissue was incubated in 30% sucrose solution overnight for cryoprotection and snap frozen afterwards. Heat-induced epitope retrieval with 0.01 M citrate buffer, pH 6.0, was performed. The following primary antibodies were used: anti-nestin (1:400, ab6320, Abcam, RRID:AB_308832), anti-CD31 (1:100, AF3628, R&D Systems, RRID:AB_2161028), anti-aquaporin 4 (1:200, ab9512, Abcam, RRID:AB_307299), anti-activated Notch1 (1:50, ab8925, Abcam, RRID:AB_306863) and anti-ki67 (1:500, ab15580, Abcam, RRID:AB_443209). Donkey anti-mouse IgG Alexa Fluor 488 (1:400; A-21202, Thermo Fisher Scientific, RRID:AB_141607), goat anti-mouse IgG Alexa Fluor 488 conjugate (1:400; A-11029, Thermo Fisher Scientific, RRID:AB_138404), donkey anti-goat IgG Alexa Fluor 633 (1:400; A-21082, Thermo Fisher Scientific, RRID:AB_141493) and donkey anti-rabbit IgG Alexa Fluor 546 (1:400; A-10040, Thermo Fisher Scientific, RRID:AB_2534016) secondary antibodies were used. Images were acquired using a Leica TCS SP5 confocal microscope.
8
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).
9
Immunostaining of Aquaporin-4, MMP-2, and Claudin-5
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The sections were incubated in 0.3% hydrogen peroxide for 30 min and in 0.1% Triton X-100 for 20 min. Then they were incubated with anti-Aquaporin-4 (AQP4) antibody (1:100; ab9512; Abcam), with anti-MMP-2 antibody (1:200; ab86607; Abcam), or with anti-claudin-5 antibody (1:200; ab15106; Abcam) overnight at 4°C and washed with PBS, incubated with secondary antibody, at 37°C for 60 min. At last, the slices were washed with PBS and sealed with the coverslip.
10
Immunofluorescent Staining of Salivary Glands
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Submandibular and lacrimal glands frozen sections were blocked with 1% BSA and 5% normal donkey serum in PBS for one hour at room temperature. The primary antibodies: AQP5 (ab78486, abcam), AQP4 (ab9512, abcam) CK5 (PRB-160P, Covance), α-SMA (ab7817, abcam), c-Kit (ab5506, abcam), Ki-67 (9129S, Cell Signaling Technology) were incubated for 24 h in 4 °C refrigerator. Polyclonal donkey anti-mouse or rabbit fluorophore-conjugated secondary antibodies in 1X PBS were applied for 1 h at room temperature. Finally, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (d1306, Invitrogen) was applied for 3 min. Images were acquired using Volocity software and the intensity was analyzed using 4–6 200× magnified fields using ImageJ. Using ImageJ software, the positive signal occupying area was calculated then divided by the total area of the tissue and a percentage was generated. An average per mouse then per group were calculated and represented as mean ± S.D.
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