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6 protocols using anti rabbit igg alexafluor 594

1

Immunohistochemical Analysis of Ion Transporters

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CFTR 596 antibody (Obtained from Cystic Fibrosis Center of North Carolina), NHE3 (Novus,#NBP1-82574), ENaC (Invitrogen #PA1-920A), Na+/K+ ATPase (Abcam #Ab7671); WGA-AlexaFluor 488 (Invitrogen #W11261) and secondary goat anti-mouse-Alexa Fluor 594 (Life Technologies #A11032), anti-mouse Alexa Fluor 488 (Life Technologies #A-11001), anti-rabbit IgG-Alexa Fluor 488 (Life Technologies #A-11008), and anti-rabbit IgG-AlexaFluor 594 (Life Technologies #A-11012) were used. Citrate buffer (DAKO#S1699) and Fluoro-gel (EMS#17985-10) were used for antigen retrieval and mounting the slides.
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2

Neuronal Marker Immunostaining Protocol

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Mouse anti-NeuN antibody (MAB377) was obtained from Millipore. Rabbit anti-VGLUT1 antibody (VGT1-3) was purchased from Mab Technologies. Chicken anti-GFP (A10262), donkey anti-mouse IgG AlexaFluor 594, anti-chicken AlexaFluor 488 and anti-rabbit IgG AlexaFluor 594 antibodies were purchased from Life Technologies. Other common reagents were from Sigma-Aldrich or Fisher Scientific.
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3

Immunofluorescence Assay for Podocyte Characterization

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Podocytes in six-well plates were xed with 4% paraformaldehyde at room temperature for 30 min, incubated in phosphate-buffered saline (PBS) containing 0.4% Triton X-100 for 10 min, and blocked with 2% bovine serum albumin at 37°C for 60 min. The cells were then incubated with the primary antibodies anti-vimentin (1:100; Boster Biological Technology, Wuhan, China) and anti-keratin (1:100; Boster Biological Technology) at 4°C overnight. PBS was used as the negative control. Then, the cells were washed with PBS and incubated with anti-mouse IgG Alexa Fluor 488 or anti-rabbit IgG Alexa Fluor 594 (1:1000; Life Technologies, Paisley, UK) for 1 h at room temperature. The glass cover slides were installed using the installation medium with 4 ,6-diamidino-2-phenylindole dye (Vector Labs, Peterborough, UK) and prepared for imaging under a uorescence microscope under ⊆200 magni cation (Olympus, Tokyo, Japan).
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4

Immunofluorescent Detection of 53BP1

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Cells were washed with PBS and fixed with 4% formaldehyde for 10 min at room temperature, permeabilized with 0.3% Triton X-100 in PBS for 5 min at room temperature and then blocked with 3% BSA/10% FBS in PBS for 1 h at room temperature. Samples were then incubated with rabbit anti-53BP1 overnight at 4°C, washed with 0.05% PBS-Tween20 and incubated with anti-rabbit IgG AlexaFluor 594 (Molecular Probes). DNA was counterstained with DAPI and images were acquired using a Zeiss AxioImager M1, using a Hamamatsu digital camera and the Volocity 4.3.2 software (Perkin Elmer).
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5

Immunofluorescent Detection of 53BP1

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Cells were washed with PBS and fixed with 4% formaldehyde for 10 min at room temperature, permeabilized with 0.3% Triton X-100 in PBS for 5 min at room temperature and then blocked with 3% BSA/10% FBS in PBS for 1 h at room temperature. Samples were then incubated with rabbit anti-53BP1 overnight at 4°C, washed with 0.05% PBS-Tween20 and incubated with anti-rabbit IgG AlexaFluor 594 (Molecular Probes). DNA was counterstained with DAPI and images were acquired using a Zeiss AxioImager M1, using a Hamamatsu digital camera and the Volocity 4.3.2 software (Perkin Elmer).
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6

Immunohistochemical Labeling of Mouse Cochlea

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Immunohistochemical labelling was carried out on fresh tissue from unexposed animals. Mouse cochleae were fixed by scali perfusion of 4% paraformaldehyde, decalcified for 14 days in 8% EDTA and then dissected for surface mount preparation or cryoprotected using sucrose and OCT before being cryosectioned at 50 μm. Nonspecific binding was blocked with 10–15% normal goat or donkey serum, 1% Triton X-100 in phosphate-buffered saline (PBS) for 1 h at room temperature (RT) and sections were then transferred to the primary antibody solution; Neurofilament 200 kDa (Sigma, rabbit, 1:5,000); peripherin (EB12405, Everest Biotech, goat, 1:10,000); CtBP2 (BD Bioscience, mouse, 1:500); vesicular acetylcholine transporter (VAChT; Phoenix, goat, 1:200) in 5–15% normal goat or donkey serum, 0.1% Triton X-100 in PBS) overnight at RT. The sections were washed in PBS and appropriate secondary antibody was applied overnight RT (Molecular Probes, 1:500 anti-rabbit IgG AlexaFluor 594, anti-goat IgG AlexaFluor 488, anti-mouse IgG AlexaFluor 488). Following a further PBS wash, DAPI was applied for 5 min at 1:5,000. Two final washes in PBS were carried out before the tissue was mounted using Vectashield (Vector Laboratories) and imaged on a Zeiss confocal microscope (Zeiss 710 NLO). Data were obtained from 1 to 3 organ of Corti z stacks (30 μm) analysed per animal.
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