After 72 h of bacterial cultivation in mineral agar media containing 20 g/L of the carbon source (glucose, fructose, propionic acid, or sunflower oil), the bacterial colonies were stained with 0.02% Sudan black solution in ethylene glycol (Sigma-Aldrich, St. Louis, MO, USA) for 30 min. Then, stained colonies were washed with 96% ethanol (Lach-Ner, Neratovice, Czech Republic). PHA producers were identified according to dark colonies and non-producing bacterial strains were decolored [58 (link),59 (link)].
Sudan black solution
Manufactured by Merck Group
Sourced in United States
Sudan black solution is a laboratory reagent used as a staining dye. It is commonly employed in histological and cytological procedures to detect the presence of lipids in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Sodium citrate tribasic dehydrate, by Merck Group (2 mentions)
Antibody diluent reagent, by Thermo Fisher Scientific (2 mentions)
Permafluor aqueous mounting medium, by Thermo Fisher Scientific (2 mentions)
Citric acid, by Merck Group (2 mentions)
Ethylene glycol, by Merck Group (1 mentions)
Ix51 inverted fluorescence microscope, by Olympus (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Antifade mountant, by Thermo Fisher Scientific (1 mentions)
Hoechst, by Merck Group (1 mentions)
Alexa fluor 594 goat anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Tricaine, by Merck Group (1 mentions)
Acridine orange, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Polysciences (1 mentions)
E3 medium, by Merck Group (1 mentions)
6 protocols using sudan black solution
1
Screening for PHA-producing Bacteria
2
Double Immunofluorescence for TREM2 and DAP12
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We also conducted double immunofluorescence to determine the relationship between TREM2 and DAP12 expression in the brain tissues. A sequential labeling procedure was applied. We first processed the immunolabeling of TREM2 with primary antibody as described earlier, followed by biotinylated anti‐goat immunoglobulin G (IgG) and streptavidin‐conjugated Alexa 488 (Molecular Probe, Life Technologies, Carlsbad, CA, USA). After completion of the first round of immunolabeling, we applied the primary antibody of DAP12 overnight, followed by 2‐hour incubation with anti‐rabbit IgG (H + L) conjugated with Alexa 568. Both fluorescence‐dye‐conjugated antibodies were used at 1:2000 from the stock provided by the manufacturer. To reduce nonspecific autofluorescence background from human brain tissues, mounted tissue sections were immersed in 1% Sudan Black solution (Sigma‐Aldrich, St. Louis, MO, USA) in 70% alcohol for 5 minutes. The excessive Sudan Black was removed by brief immersion of the slides in 70% alcohol and distilled water before coverslipping 26 . Slides were coverslipped with Vectashield (Vector Laboratories, Burlingame, CA, USA). The images were captured with a charge‐coupled device (CCD) camera attached to the Olympus IX51 inverted fluorescence microscope (Olympus, Center Valley, PA, USA). Image overlay was composed by the Olympus software DP Controller version 3.2.1.276.
3
Immunofluorescence Staining of Paraffin Tissue Sections
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Paraffin tissue slices were deparaffinized in 3 × 5 min washes of xylene and rehydrated in 2 × 10 min washes of 100% ethanol and 95% ethanol. Tissue slices were subjected to antigen retrieval using heated Citrate buffer (10 mm Sodium Citrate, pH 6) for 10 min, permeabilized in permeabilization buffer (0.2% Triton X‐100 in TBST) for 10 min then washed 2 × 5 min with TBST. Slides were rinsed with 1× PBS and incubated with Sudan black solution (Sigma) for 30 min. Following 3× dipping in 70% ethanol, slides were rinsed 4× in 1× PBS and blocked in 10% normal goat serum with 1% bovine serum albumin (BSA) for 1.5 h at RT. Subsequently, primary antibody was added and incubated overnight in a humidified chamber at 4 °C. The next day, slides were rinsed twice for 5 min in 1× PBS and stained with secondary antibodies Alexa Fluor 488 goat anti‐mouse (1:500, ThermoFisher), Alexa Fluor 594 goat anti‐rabbit secondary antibody (1:500, ThermoFisher), and Hoechst (1:5000, Sigma‐Aldrich). Following a 2 h incubation at RT, slides were mounted with antifade mountant (ThermoFisher) before being sealed. Images were captured using a fluorescent tissue microscope (Nikon), and processed using NIS‐Elements Software.
4
Immunofluorescence Tissue Staining Protocol
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Transverse myocardial tissue sections were heated at 80 °C for 30 min, deparaffinized with xylene, and stepwise rehydrated by successive rinsing in decreasing concentrations of ethanol (100, 96, 90, and 80%) prior to placing in deionized water. Antigen retrieval was then performed by submerging tissue sections in citrate retrieval buffer [2.4 g/L sodium citrate tribasic dehydrate (S4641, Sigma-Aldrich), 0.35 g/L citric acid (C0759, Sigma-Aldrich), pH 6.0] for 10 min at approximately 100 °C. Sections were allowed to cool for 10 min on ice and washed in deionized water prior to application of diluted antibodies. Primary and secondary antibodies were diluted in antibody diluent reagent (003218, Invitrogen) prior to use. All sections were counterstained with 1 µg/mL DAPI (D3571, Invitrogen) for 10 min and subsequently incubated with 0.1% Sudan Black solution (199664, Sigma-Aldrich) in 70% ethanol for 15 min to mitigate the autofluorescence signal. Sections were then washed with 1 × PBS (3 times; 3 min each), rinsed in deionized water, and finally mounted under glass coverslips using PermaFluor Aqueous Mounting Medium (TA-030-FM, ThermoFisher Scientific). Images were acquired digitally using a Nikon Eclipse Ti fluorescence microscope and analyzed using NIH Image J software (1.46r).
5
Immunostaining of Myocardial Tissue Sections
6
Lipid Staining and Fluorescent Imaging of Embryos
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For Sudan black staining, embryos were fixed with 4% PFA (Polysciences) in PBS for 2 h at room temperature, rinsed in PBS, and incubated in Sudan black solution (Sigma-Aldrich) as described previously (Sheehan and Storey, 1947 (link); Le Guyader et al., 2008 (link)). For acridine orange staining, zebrafish larvae were anesthetized with Tricaine (Sigma-Aldrich), incubated in a solution of 3 µg/ml acridine orange (Sigma-Aldrich) in E3 medium with Tricaine (Sigma-Aldrich) for 30 min, washed twice in E3 with Tricaine, and then analyzed under a fluorescent stereomicroscope.
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