After 14 days of culture, samples were fixed, permeabilized and blocked using the same method used to prepared cells for F-actin staining. For immunocytochemical staining, samples were then incubated for 18 h at 4°C in a range of antibodies, anti-ALDH3A1 1:50 (ab76976; Abcam, Cambridge, United Kingdom), anti-keratocan 1:50 (sc-66941; Santa Cruz, Heidelberg, Germany), or anti-α-smooth muscle actin (αSMA) 1:50 (ab7817; Abcam). They were then washed thrice with PBS, 20 min for every wash and then incubated with fluorescently labelled antibodies for 1h at ambient temperature, in the dark. To highlight ALDH3A1 and Keratocan, Donkey anti-rabbit Alexa Fluor 488 (ab150073; Abcam) was used. For αSMA, goat anti-mouse biotin followed by ExtrAvidin-FITC (B7151 and E2761; Sigma). Constructs were washed with PBS and counterstained with DAPI (1 mg/mL,1:500 dilution) to visualize nuclei. Confocal microscopy was performed on the Leica SP8 with LAS X Advanced software 2.13 post-processing.
Ab150073
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab150073 is a monoclonal antibody that binds to a specific target. The core function of this product is for use in various laboratory applications, such as immunoassays and immunohistochemistry. No further details are provided to maintain an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab104139, by Abcam (12 mentions)
Lsm 700, by Zeiss (9 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (7 mentions)
Ab7260, by Abcam (6 mentions)
Ab150108, by Abcam (6 mentions)
Ab150107, by Abcam (6 mentions)
Lsm 780 confocal microscope, by Zeiss (6 mentions)
Ab150075, by Abcam (5 mentions)
Ab150064, by Abcam (5 mentions)
Ab150105, by Abcam (5 mentions)
Ab7475, by Abcam (5 mentions)
Ab175472, by Abcam (4 mentions)
Ab150129, by Abcam (4 mentions)
Ab150131, by Abcam (4 mentions)
Ab8295, by Abcam (4 mentions)
Lsm 710, by Zeiss (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Ab76976, by Abcam (3 mentions)
Ab7817, by Abcam (3 mentions)
Sc 66941, by Santa Cruz Biotechnology (3 mentions)
142 protocols using ab150073
1
Immunocytochemical Characterization of Corneal Cells
2
Multicolor Immunofluorescence Imaging
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Immunohistochemistry protocol was based on those described earlier (Marathe et al., 2015; (link)Yanpallewar et al., 2010) (link). In brief, the sections were removed from the freezing mixture and washed three times with PB. Sections were then blocked for 1 hr with 10% normal donkey serum +3% bovine serum albumin (BSA) +0.3% TritonX100 in 0.1-M PB. Sections were incubated overnight at 4°C with primary antibodies (Goat pAb anti-SOX9, 1:200; R&D systems, AF3075; chicken pAb anti-GFAP, 1:1,000, Novus, NBP1-05198 and rabbit pAb anti-S100β, 1:1,000, Synaptic systems, 287003). On the second day, the sections were washed three times with PB and incubated for 2 hr at room temperature with secondary antibodies (donkey anti goat 568, abcam, ab175474; goat anti chicken Alexa Flour 594, abcam, ab150172; and donkey anti rabbit Alexa Flour 488, abcam, ab150073). After incubation, sections were washed three times in PB and were mounted on a glass slide in the mounting medium with DAPI (Abcam ab104139). The slides were imaged on a Zeiss LSM 880 airyscan confocal microscope with a Plan-Apochromat 20X/0.8 M27 objective. 1024X1024 pixels, 8-bit images were acquired at 0.860-µm z-step size.
3
Immunofluorescence Analysis of Complement Deposition
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Mice were anesthetized with pentobarbital sodium (50 mg/kg, i.p.) and lung tissues were excised and fixed with 10% neutral buffered formalin. Paraffin-embedded sections were incubated with rabbit anti-C4d IgG (1:200, overnight at 4°C; Hycult Biotech, HP8033) or rabbit anti–C5b-9 IgG (Bioss Antibodies, bs-2673R) and then successively reacted with donkey anti–rabbit IgG H&L (Alexa Fluor 488) (1:500, 1 hour at room temperature; Abcam, ab150073). For LRA deposition, paraffin-embedded sections were incubated directly with with donkey anti–rabbit IgG H&L (Alexa Fluor 488) as above. Nuclei were stained with Hoechst 33342 (MilliporeSigma). Confocal images were acquired using a Zeiss Axio Imager Z2 with ApoTome.2 microscope equipped with Axiocam 503 Mono, X-Cite 120 LED Boost System and Zen 2.3 software (Carl Zeiss).
4
Immunohistochemical Detection of VEGF and Amyloid-β
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This procedure was performed by a standard indirect method as described previously [8 (link)]. Primary antibodies and dilutions were as follows: anti-VEGF (1:250; # ab14078, Abcam) and anti-amyloid-β1–42 (1:250; # ab10148, Abcam). After incubation with the respective secondary antibodies conjugated with Alexa Fluor 555 or Alexa Fluor 488 (## ab150170, ab150073, Abcam) diluted 1:250, the tissue slices were coverslipped with the Fluoro-shield mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI; # ab104139, Abcam) and examined under an Axioplan 2 microscope (Zeiss).
5
Immunohistochemical Analysis of Reelin and DAB1
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Kidney tissues were dissected and fixed in 4% paraformaldehyde in phosphate buffer and dehydrated in 100% ethanol. They were embedded in paraffin wax, serially sectioned at 5 μm, and mounted on glass slides. The sections were deparaffinized in xylene and rehydrated in ethanol and water as we described previously (21 (link)-23 (link)). After washing in phosphate-buffered saline (PBS), the sections were incubated in humid chamber over night with primary antibodies: Mouse Monoclonal Reelin antibody (1:70 dilutions; sc-25346, Santa Cruz Biotechnology, Dallas, TX, USA), Rabbit Polyclonal Anti-DAB1 (phospho Y232) antibody (1:400 dilutions; ab 78200, Abcam, Cambridge, UK). After washing in PBS, secondary antibodies, Donkey Anti-Rabbit IgG H&L, Alexa Fluor 488 (dilution 1:400, ab 150073, Abcam) and Goat Anti-Mouse IgG H&L, TRITC (dilution 1:400, ab 6786, Abcam) were applied for one hour. The nuclei were stained with DAPI for 2 minutes, washed in PBS, and coverslipped. Controls for specificity of staining, with omission of the primary antibody, were included in the staining procedure to exclude nonspecific staining.
6
Immunohistochemical Analysis of TRPV4 in Rat Cerebral Arteries
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FHH rat cerebral arterial segments were fixed in 4% paraformaldehyde for 24 h and then paraffin embedded. The embedded arterial segments were sectioned at 4 μM thickness and dried on glass slides at 45°C overnight. The sectioned samples were antigen retrieved with DAKO target retrieval solution (pH = 6, DakoCytomation, s1699) for 40 min at 99°C followed by cool down at room temperature for 20 min. The slides were then rinsed with TBST and protein blocked for 30 min with the reagent removed by blow off. Polyclonal anti-TRPV4 antibody (Alomone ACC-034) was applied to the slides at a dilution of 1:200 and incubated overnight at 4°C. The slides were then rinsed twice with TBST for 5 min and treated with a secondary antibody, Donkey anti-Rabbit Alexa 488 (ab150073, Abcam), at a dilution of 1:750 for 45 min at room temperature. The slides were then rinsed twice with TBST for 5 min and counterstained with DAPI at a dilution of 1:1000 for 10 min followed by a rinse with distilled water and covered with ProLong Gold antifade mounting medium (Life Technologies) for 30 min and sealed with nail polish. Images were captured with AIM 4.2 software controlling Zeiss LSM510 microscope (Jena, Germany) and 40 x objectives.
7
Immunofluorescent Staining of Beta-Catenin
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HUVECs were fixed with 4% formaldehyde and incubated with PBS containing 0.1% Triton X-100 (PBS-BT) and 5% normal serum for 1 h at room temperature. The solution was then removed and replaced with a solution of β-catenin (ab325728, Abcam) diluted 1 : 200. After incubation for 2 h at room temperature, the samples were rinsed and further incubated for 1 h with donkey anti-rabbit IgG H&L (Alexa Fluor® 488) (ab150073, Abcam) diluted 1 : 250. Cells were rinsed in PBST, followed by the addition of 1 μg/mL DAPI (62248, Thermo Scientific). Samples were visualized using a Nikon Ts2-FL microscope (Nikon, Japan).
8
Glial Cell Marker Colocalization Analysis
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A single brain section from each animal of each was group were co-stained with
IBA1 and PBR. In addition, we also co-stained glial fibrillary acidic protein
(GFAP) and PBR. Primary antibodies included: Anti-PBR (goat polyclonal primary
antibody, 1:50; Abcam Cat# ab118913, RRID: AB_10898989), GFAP (rabbit polyclonal
antibody, 1:500; Abcam Cat# ab7260, RRID: AB_305808), and IBA-1 (rabbit
polyclonal primary antibody, 1:500; Wako Chemicals USA, Cat# 019-19741, RRID:
AB_2313566). Secondary fluorescent antibody products included: Alexa Fluor® 488
(donkey polyclonal secondary antibody to rabbit IgG H&L, 1:500; Abcam Cat#
ab150073, RRID: AB_2636877) and Alexa Fluor® 568 (donkey polyclonal secondary
antibody to goat IgG H&L, 1:500; Abcam Cat# ab175704). Antibody staining
combinations included: IBA1/PBR and GFAP/PBR. All photomicrographs were taken
blinded and the analyses were done blinded as well.
IBA1 and PBR. In addition, we also co-stained glial fibrillary acidic protein
(GFAP) and PBR. Primary antibodies included: Anti-PBR (goat polyclonal primary
antibody, 1:50; Abcam Cat# ab118913, RRID: AB_10898989), GFAP (rabbit polyclonal
antibody, 1:500; Abcam Cat# ab7260, RRID: AB_305808), and IBA-1 (rabbit
polyclonal primary antibody, 1:500; Wako Chemicals USA, Cat# 019-19741, RRID:
AB_2313566). Secondary fluorescent antibody products included: Alexa Fluor® 488
(donkey polyclonal secondary antibody to rabbit IgG H&L, 1:500; Abcam Cat#
ab150073, RRID: AB_2636877) and Alexa Fluor® 568 (donkey polyclonal secondary
antibody to goat IgG H&L, 1:500; Abcam Cat# ab175704). Antibody staining
combinations included: IBA1/PBR and GFAP/PBR. All photomicrographs were taken
blinded and the analyses were done blinded as well.
9
MALAT1 Expression and KHSRP Localization
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The MALAT1 FISH probes were designed and supplied by RiboBio (Guangzhou, China). According to manufacturer’s instructions of FISH kit (RiboBio), cells were harvested and fixed with 4% paraformaldehyde. Then, cells were treated with 0.5% Triton X-100, and followed by incubated with the specific MALAT1 probe (1: 50) for overnight. Next, the cells were incubated with anti-KHSRP (1:200, ab150393, Abcam) for overnight, followed by the incubation with Alexa fluor 488-conjugated secondary antibody (ab150073, Abcam, Cambridge, UK) for 2 h. Cells were further stained with 4′, 6-diamidino-2-phenylindole (DAPI) to label cell nucleus. Finally, cells were observed and photographed under a confocal microscope (Olympus, Japan).
10
Hypothalamic Gene Expression in Mice
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Twenty-four mice (male, 8 weeks old) feeding on a standard chow diet were equally divided into eight groups (NC-0h, NC-1h, NC-2h, NC-3h; D3-0h, D3-1h, D3-2h, D3-3h). For gene expression assays, the hypothalamus was harvested 0–3 hours following D3 administration (NC: saline), and the expression levels of c-Fos, AgRp and Pomc were quantified by quantitative RT-PCR.
The same experiment was carried out for the immunofluorescence. c-Fos staining was performed as described in a previous study.66 (link) Briefly, mice were anaesthetised with a lethal dose of pentobarbital and transcardially perfused with PBS followed by 4% paraformaldehyde. Brains were removed, placed in 4% paraformaldehyde overnight and dehydrated in 30% sucrose for 1 week. Brains were cut into 25 mm sections. The sections were treated as described above and incubated overnight at room temperature in mouse anti-FOS (1:500; ab208942; Abcam) or rabbit anti-POMC (1:100; ab254257; Abcam). Detection and labelling were performed using secondary antibodies conjugated to Alexa Fluor-594 donkey anti-mouse IgG (H+L) (1/500, ab150108, Abcam) or Alexa Fluor-488 donkey anti-rabbit IgG (H+L) (1/500, ab150073, Abcam), and imaging was performed as described above. Images were pseudocoloured using Photoshop software (Adobe) or ImageJ (NIH).
The same experiment was carried out for the immunofluorescence. c-Fos staining was performed as described in a previous study.66 (link) Briefly, mice were anaesthetised with a lethal dose of pentobarbital and transcardially perfused with PBS followed by 4% paraformaldehyde. Brains were removed, placed in 4% paraformaldehyde overnight and dehydrated in 30% sucrose for 1 week. Brains were cut into 25 mm sections. The sections were treated as described above and incubated overnight at room temperature in mouse anti-FOS (1:500; ab208942; Abcam) or rabbit anti-POMC (1:100; ab254257; Abcam). Detection and labelling were performed using secondary antibodies conjugated to Alexa Fluor-594 donkey anti-mouse IgG (H+L) (1/500, ab150108, Abcam) or Alexa Fluor-488 donkey anti-rabbit IgG (H+L) (1/500, ab150073, Abcam), and imaging was performed as described above. Images were pseudocoloured using Photoshop software (Adobe) or ImageJ (NIH).
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