Previously reported protocols were applied (5 (link), 6 (link)). To identify podocytes, we used a combination of WT1 (Agilent Technologies; IS05530-2) and DACH1 (Sigma-Aldrich; HPA012672) (32 (link)) as primary antibodies; Alexa-Fluor 488, -555, and/or -647 as secondary antibodies (Invitrogen; A21202, A31572, A31571, and A31573, respectively) depending on the experiment; and a DNA marker to identify single nuclei — either DAPI (Sigma-Aldrich; D9542) or DRAQ5 (Abcam; ab108410). Optical images were obtained using inverted laser confocal microscopes (Nikon and LSM800, Zeiss), stored in 1024 × 1024 pixel frames. Each image contained 1 glomerulus.
D9542
Manufactured by Abcam
Sourced in United States, Israel
D9542 is a reagent used in laboratory settings. It is a chemical compound that serves a specific function in research or analysis procedures. The core purpose of D9542 is to facilitate certain laboratory processes, but a detailed description of its intended use cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Ab108410, by Abcam (1 mentions)
A31572, by Thermo Fisher Scientific (1 mentions)
Hpa012672, by Merck Group (1 mentions)
A31571, by Merck Group (1 mentions)
A31573, by Merck Group (1 mentions)
A21202, by Thermo Fisher Scientific (1 mentions)
Lsm 800, by Zeiss (1 mentions)
P6148, by Merck Group (1 mentions)
Ab14083, by Abcam (1 mentions)
Ab150107, by Thermo Fisher Scientific (1 mentions)
Ab150073, by Thermo Fisher Scientific (1 mentions)
Ab150075, by Abcam (1 mentions)
Anti cofilin, by Abcam (1 mentions)
Fm1 43, by Thermo Fisher Scientific (1 mentions)
Anti igg1, by BD (1 mentions)
Phalloidin, by Thermo Fisher Scientific (1 mentions)
Anti prestin, by Santa Cruz Biotechnology (1 mentions)
Anti sox2, by Santa Cruz Biotechnology (1 mentions)
Ab176753, by Abcam (1 mentions)
4 protocols using d9542
1
Podocyte Identification Protocol
2
Immunofluorescence Staining Protocol
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Immunofluorescence analysis was done according
to standard protocols. In brief, we started with sample
fixation in 4% paraformaldehyde (PFA, Sigma-Aldrich,
USA, P6148) for 20 minutes at room temperature (RT),
then, permeabilization using 0.1% Triton X-100 for 10
minutes. The samples were then incubated in blocking
solution (10% secondary antibodies host serum) for 1
hour at RT, followed by an overnight incubation with
primary antibodies at 4˚C. Next, the cells were washed
in phosphate buffered saline (BSA) and incubated with
secondary antibodies for 45 minutes in an incubator
at 37˚C temperature.Table S1 (See Supplementary
Online Information atwww.celljournal.org ) lists the
primary and secondary antibodies used in this work. As
negative control we incubated the cells with secondary
antibodies only after the permeabilization step. Nuclei
were stained by incubating the samples in 4, 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, USA, D9542, 5
μg/ml) or propidium iodide (Abcam, UK, ab14083) in
PBS for 3 minutes at RT. The analysis was done under a
fluorescent microscope (Olympus, Japan, IX71).
to standard protocols. In brief, we started with sample
fixation in 4% paraformaldehyde (PFA, Sigma-Aldrich,
USA, P6148) for 20 minutes at room temperature (RT),
then, permeabilization using 0.1% Triton X-100 for 10
minutes. The samples were then incubated in blocking
solution (10% secondary antibodies host serum) for 1
hour at RT, followed by an overnight incubation with
primary antibodies at 4˚C. Next, the cells were washed
in phosphate buffered saline (BSA) and incubated with
secondary antibodies for 45 minutes in an incubator
at 37˚C temperature.
Online Information at
primary and secondary antibodies used in this work. As
negative control we incubated the cells with secondary
antibodies only after the permeabilization step. Nuclei
were stained by incubating the samples in 4, 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, USA, D9542, 5
μg/ml) or propidium iodide (Abcam, UK, ab14083) in
PBS for 3 minutes at RT. The analysis was done under a
fluorescent microscope (Olympus, Japan, IX71).
3
Immunostaining Cochlear Sensory Epithelia
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The cochleae were placed in the medium containing 3 mM FM1-43 (Thermo Fisher, F35355) for 90 s and washed three times in PBS (pH 7.2). The cochleae then were dissected and fixed with 4% polyoxymethylene for 1 h and permeabilized with 0.5% Triton X-100 for 1 hour. The sensory epithelia were then incubated with the following primary antibodies overnight at 4 °C: anti-LIMK1 (Santa Cruz, sc-8387); anti-LIMK2 (Santa Cruz, sc-5577); anti-MYO7A (Proteus Bioscience, 25-6790); anti-SOX2 (Santa Cruz, sc-17320, sc-365823); anti-CtBP2 (C-terminal-binding protein 2), anti-IgG1 (BD Biosciences, 612044), anti-PSD95 IgG2a (Millipore, MAB1596), anti-cofilin (Abcam, ab42824), anti p-cofilin (Santa Cruz, sc-12912), and anti-prestin (Santa Cruz, sc-22692). Phalloidin (Invitrogen, A34055) was used to stain the actin cytoskeleton, and DAPI was used to label the nuclei. The tissues were washed three times with PBST (0.1 M phosphate buffer, pH 7.2, with 0.1% Triton X-100) and incubated for 1 h (37 °C) with DAPI (Sigma-Aldrich, D9542) and suitable secondary antibody (Abcam: ab150075, ab150105, ab150074, ab150073, ab150107; Invitrogen: A21131, A21124). Finally, the sensory epithelia were mounted on glass slides with Fluoromount-G mounting medium. Images were taken using a Zeiss LSM700 confocal microscope.
4
Immunofluorescence Staining of Cytoskeleton and Mucins
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Immunofluorescence stains were used for model validation and the quantitative evaluation of cytoskeletal F-actin alterations and MUC1 secretion. The following specific antibodies were used (i) 1:500 dilution of phalloidin-iFluor 488 (Ab 176753, Abcam, Cambridge, UK), (ii) 1:50 dilution of anti-MUC1 (VU4H5) Alexa Fluor® 680 antibody (sc-7313 AF680, ENCO, Petah Tikva, Israel), (iii) 1:50 dilution of anti-MUC16 (C-6) Alexa Fluor® 594 (sc-365002 AF594, ENCO, Petah Tikva, Israel) and (iv) DAPI (D9542, Abcam) for the nuclei. The HCEC controls and those obtained immediately after exposure to WSS were fixed by incubation in 4% paraformaldehyde (PFA) for 10 min at RT. The cells were then incubated overnight at 4 °C with DPBS 1% BSA and the specific antibodies. The HCEC were washed three times with DPBS for 5 min after each step in the shaker machine. Following staining, the cultured cells on the well bottom were imaged under a Leica SP8 confocal microscope with a 20×/0.75 dry lens.
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