The slides were heated and subsequently put in xylene, washed with gradient alcohol for deparaffinization, and then rehydrated with distilled water. After that, antigen retrieval was performed in 1 mM boiling EDTA buffer (pH 8.0; EMD Millipore) for 15 min at 92–100°C. The slides were incubated with 3% hydrogen peroxide for 30 min, washed, and blocked with 5% goat serum (EMD Millipore) for 60 min at room temperature. A mouse monoclonal anti-LASS2 antibody (cat. no. sc-390745; dilution, 1:100, Santa Cruz Biotechnology, Inc.) was applied to each slide overnight at 4°C. Following washing with PBS (pH 7.4), the slides were incubated for 30 min with a rabbit anti-mouse secondary antibody (cat. no. A-11062, dilution, 1:400; Invitrogen; Thermo Fisher Scientific, Inc.). Subsequently, the slides were stained with diaminobenzidine, counterstained with hematoxylin, dehydrated in ethanol, cleared in xylene, mounted on coverslips and examined under an optical microscope (Eclipse E200; Nikon Corporation, Tokyo, Japan). The cells with yellow-brown staining in their cytoplasm were considered LASS2-positive. The mean density of staining, calculated using Image Pro-Plus (Media Cybernetics, Inc., Rockville, MD, USA) as previously described (16 (link)), was used to quantify the LASS2 staining.
Edta buffer
Manufactured by Merck Group
Sourced in United States
EDTA buffer is a chemical solution used in various laboratory applications. It serves as a chelating agent, binding to certain metal ions and preventing their interference in analytical processes. The core function of EDTA buffer is to maintain the stability and integrity of samples during analysis and experimentation.
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Lab products found in correlation
Dibutylphthalate polystyrene xylene (dpx), by Merck Group (2 mentions)
Succinic acid, by Merck Group (2 mentions)
Agilent 8453 spectrophotometer, by Agilent Technologies (2 mentions)
Myoglobin mb from equine heart, by Merck Group (2 mentions)
Pt module, by Lab Vision (2 mentions)
Image pro plus, by Media Cybernetics (1 mentions)
Eclipse e200, by Nikon (1 mentions)
Ultra 5 block, by Thermo Fisher Scientific (1 mentions)
Bx51 light microscope, by Olympus (1 mentions)
Goat anti polyvalent solution, by Thermo Fisher Scientific (1 mentions)
Eclipse ti microscope, by Nikon (1 mentions)
Chondroitin sulfate sodium salt from shark cartilage, by Merck Group (1 mentions)
Pepstatin a, by Merck Group (1 mentions)
Dna sodium salt from calf thymus, by Merck Group (1 mentions)
Potassium dihydrogen phosphate, by Merck Group (1 mentions)
Ab2302, by Merck Group (1 mentions)
Fluoroshield with dapi, by Merck Group (1 mentions)
Nis element, by Nikon (1 mentions)
Alizarin red staining, by Merck Group (1 mentions)
Anti baff r, by Abcam (1 mentions)
17 protocols using edta buffer
1
Immunohistochemical Analysis of LASS2 Expression
2
Immunohistochemical Analysis of Testicular Apoptosis
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Slides for immunohistochemistry, were rehydrated and quenched in 3% H 2 O 2 (Thermo Scientific, Fremont, CA, USA). Heat-activated antigen retrieval was performed in Tris-ethylenediaminetetraacetic acid (EDTA) buffer (Merck) at pH 6.0. Non-specific binding was eliminated via Ultra V Block (Thermo Scientific). One of the sections in each slide was stained with rabbit active caspase-3 antibody (Abcam, Cambridge, MA. USA. Cat. # ab44976), while second section was stained with goat anti-polyvalent solution (Thermo) instead of primary antibody as control for non-specific reactions. Tissues were incubated with primary antibodies at +4°C overnight within a humidified chamber. goat anti-polyvalent solution (Thermo Scientific, Waltham, MA, USA) was used as secondary antibody. 3,3′-Diaminobenzidine (DAB) staining was performed with UltraVision Plus Large Volume Detection Kit (Thermo Scientific), and slides were counterstained with haematoxylin. Tubules examined under Olympus BX51 light microscope (Olympus Optical Co., Ltd., Tokyo, Japan) were evaluated according to semiquantitative method (McCarty et al. 1986) , which is adapted to testes slides (Gunduz et al. 2009 ). The number of counted spermatogenic cells was increased to 100 for each animal for the study. Caspase-3-positive cells per 100 spermatogenic cells were determined.
3
Immunohistochemistry of Tyrosine Hydroxylase in Hypertensive Mouse Kidneys
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We performed immunohistochemistry in the kidneys of our normotensive BPN/3J mice and hypertensive BPH/2J mice. Kidney tissues from both our BPN/3J and BPH/2J mice were sectioned at 5 μm onto positively charged microscope slides and de-waxed in xylene and rehydrated in ethanol. Antigen retrieval was performed on the slides by heating in EDTA buffer (pH 8.5; Sigma-Aldrich, Sydney, NSW, Australia). Slides were treated with 3% hydrogen peroxide and then blocked in 5% FCS in PBS/0.1% Tween-20. Tyrosine hydroxylase was detected with rabbit anti-tyrosine hydroxylase antibody (AB152; Merck Millipore, Melbourne, VIC, Australia). Antibody binding was detected with anti-rabbit (1:100, Santa Cruz Biotechnology, Sydney, NSW, Australia) secondary antibodies conjugated to HRP, followed by treatment with diaminobenzidine (DAB, Ventana, AZ, USA). Tissues were counterstained with haematoxylin before being dehydrated in ethanol and cleared in xylene and mounted with DPX (Sigma-Aldrich, Sydney, NSW, Australia). Photomicrographs were taken of stained kidneys from mice using a Nikon Eclipse Ti Microscope (Nikon Instruments Inc, Tokyo, Japan). Tyrosine hydroxylase positive nerves were counted in random fields of view.
4
Quantifying DNA and GAG in Chondrogenesis
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Pellets were digested at day 21 of chondrogenic differentiation using 1 mg/mL Proteinase K, 1 mM iodoacetamide, 10 μg/mL Pepstatin A in 50 mM Tris, and 1 mM EDTA buffer (pH 7.6; all Sigma-Aldrich) for 16 h at 56°C, followed by Proteinase K inactivation at 100°C for 10 min. Afterward, to determine the amount of DNA, cell lysates were treated with 0.415 IU heparin and 1.25 µg RNase for 30 min at 37°C, followed by the addition of 30 μL CYQUANT GR solution (Invitrogen). The samples were analyzed using a SpectraMax Gemini plate reader with an excitation of 480 nm and an emission of 520 nm. As a standard, DNA sodium salt from calf thymus (Sigma-Aldrich) was used. To determine the amount of GAG, cell lysates were incubated with 1, 9-dimethylmethylene blue (DMB), as previously described by Farndale et al. (1986 (link)), and analyzed with an excitation of 590 nm and 530 nm. The 530:590 nm ratio was used to determine the GAG concentration. As a standard, chondroitin sulfate sodium salt from shark cartilage (Sigma-Aldrich) was used.
5
Sulfheme Complex Formation in Mb and rHbI
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Myoglobin (Mb) from equine heart was purchased from Sigma-Aldrich. Recombinant HbI (rHbI) was prepared and purified as previously reported [23] (link) and used as control. Both proteins were dissolved in a 100 mM Succinic Acid, 100 mM Potassium dihydrogen phosphate, and 1 mM EDTA buffer, 6.5 pH (all purchased in Sigma-Aldrich). The oxy-derivatives were prepared by adding [1:15] concentration ratio of [protein: sodium dithionite] under anaerobic conditions followed by O2 purging [4] (link). The H2S solution was prepared by dissolving sodium sulfide (purchased in Alfa-Aesar) in the previously mention anaerobic buffer. The sulfheme complex formation was monitored through its characteristic 620 nm band by UV–vis spectroscopy using an Agilent 8453 spectrophotometer [8] (link). The sulfheme complex was acquired by adding H2S to the oxyMb complex in a [1:70] concentration ratio of [oxyMb: H2S] that provides the highest intensity and stability of the 620 nm characteristic band.
6
Sulfheme Complex Formation in Myoglobin
7
Immunofluorescence Staining of Bone Markers
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After deparaffinization and rehydration, samples were subjected to heat-induced antigen retrieval in EDTA buffer, pH 8.5 (Sigma-Aldrich). Samples were permeabilized with 0.1% Triton X-100 for 10 min and blocked with 3% BSA (w/v) for 1 h at room temperature. Samples were stained with rabbit anti-human sclerostin (1:10, ab75914), rabbit anti-human ALP (1:10, ab75699), rabbit anti-human active caspase-3 (1:100, ab2302), rabbit anti-human FGF23 (1:50, ab192497), or rabbit anti-human Dkk-1 (1:50, ab61034) overnight at 4 °C, followed by incubation with a secondary stain (1:100 TRITC-conjugated goat anti-rabbit IgG, ab50598) for 1 h at room temperature and counterstained with DAPI containing mounting medium (Fluoroshield with DAPI, Sigma). To identify PCa cells, samples were stained with mouse anti-human pan cytokeratin (1:100, ab86734) followed by secondary staining with Alexa-Fluor 488-conjugated goat anti-mouse IgG (1:100, ab150113). All antibodies were purchased from Abcam.
Fluorescence was quantified using image analysis software (NIS-Elements, Nikon). For each sample, 10 regions of interest (ROI) were randomly selected, each containing 8–10 cells. Mean intensity was quantified for each ROI and averaged for all samples of the same group.
Fluorescence was quantified using image analysis software (NIS-Elements, Nikon). For each sample, 10 regions of interest (ROI) were randomly selected, each containing 8–10 cells. Mean intensity was quantified for each ROI and averaged for all samples of the same group.
8
CMV DNA Quantification Protocol
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Standard Reference Material 2366 Cytomegalovirus (CMV) for DNA Measurements, composed of HCMV Towne∆147 bacterial artificial chromosome DNA, was purchased from the National Institute of Standards and Technology (USA) [21 (link)]. Initially, 150 μL component C (declared concentration, 19,641 copies per microlitre) was diluted in 1.35 mL 1× tris(hydroxymethyl)aminomethane–EDTA buffer (Sigma-Aldrich, USA), and aliquoted and stored at −80 °C [7 (link)]. As the National Institute of Standards and Technology material is composed of purified plasmid DNA, no further DNA extraction was needed before its amplification. Because of plasmid instability reports from the manufacturer, aliquots were tested for the concentration and stability on the QX100 system by means of the UL54 assay (UL54 is a DNA polymerase gene of HCMV). The mean aliquot concentration (± standard error) was estimated as 1050 (±30) copies per microlitre (henceforth indicated as the nominal concentration).
9
Multiplex Immunofluorescence Tissue Staining
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Whole-tissue section slides were de-paraffinized and subjected to antigen retrieval using EDTA buffer (Sigma-Aldrich) pH=8.0 and boiled for 1 hour at 96°C in a pressure-boiling container (PT Module, Lab Vision). The sections were blocked for 10 min at room temperature. The sections were incubated with primary antibodies against Gal-1 (CST, 13888), KLF12 (Bioss, bs-16783R), PD-1 (CST, 84651S), PD-1 (CST, 86163), CD8 (Abcam, 217344), and CD8 (CST, 85336) for 1 hour at room temperature. Staining was performed consecutively, and each marker was detected before the application of the next antibody. According to the manufacturer’s instructions, the primary antibody was detected using the Opal Polymer HRP Ms+ Rb detection reagent, and Opal 5-Color Manual IHC was performed with four reactive fluorophores: Opal 520, Opal 570, Opal 620, and Opal 650 plus DAPI nuclear counterstain (Panovue, PerkinElmer).
10
Histological and Immunostaining Analysis of Aortic Valve
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For histology and immunostaining, 8 µm sections were cut using a microtome (SLEE medial, Mainz, Germany). Russell-Movat pentachrome (Abcam, Cambridge, UK) or Alizarin Red staining (Sigma Aldrich, St. Louis, MO, USA) were performed according to the manufacturer’s instructions. For immunohistochemistry analysis, after antigen retrieval with EDTA buffer (Sigma-Aldrich, St. Louis, MO, USA), slices were incubated overnight with an anti-BAFF-R or CD138 antibodies (Abcam, Cambridge, UK) diluted at 1:250 or 1:8,000, respectively. Slides were stained with HRP/DAB detection kit (Abcam, Cambridge, UK) and counterstained with hematoxylin to visualize cell nuclei. Images of entire aortic valve cusps were composed using NIS-Elements Microscope Imaging Software (Nikon, Tokyo, Japan) from a series of adjacent pictures taken with a 4 × objective taken with a Nikon Eclipse Ni microscope (Nikon, Tokyo, Japan). Immunostaining images were taken with a 20 × objective with a Nikon Eclipse Ni microscope (Nikon, Tokyo, Japan).
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