The transgenic strain CF1407 daf-16 (mu86) I; muIs 71 [pKL99(daf-16Ap::GFP::daf-16A(bKO)) + pRF4(rol-6)] was used to detect the intracellular localization of GFP-tagged DAF-16 protein. Randomly selected 4-day-old worms that were fed on plates lawned with E. coli OP50 or Weissella species for 24 h were washed three times with M9 buffer. Worms were then placed onto 5% agar pads coated with 10 mM sodium azide (Junsei Chemical, Tokyo, Japan) in M9 buffer to induce anaesthesia. GFP images were taken using green excitation light (460− 495 nm), a GFP (green fluorescence protein) channel of a laser confocal scanning microscope (Olympus Ix81-FV1000, Japan). Localization of DAF-16 GFP from the cytoplasm into the cell nuclei was examined by analysing the degree of nuclear GFP fluorescence. Three different states can easily be distinguished (no, weak, or strong nuclear GFP fluorescence), which are related to a cytoplasmic, intermediate, or nuclear location of DAF-16-GFP60 (link). The degree of nuclear translocation of DAF-16 was evaluated by counting the number of worms showing either weak or strong nuclear GFP fluorescence. Three independent experiments were performed with over 90 worms for each bacterial species.
Sodium azide
Manufactured by Junsei
Sourced in Japan
Sodium azide is a chemical compound commonly used as a laboratory reagent. It is a white crystalline solid with the chemical formula NaN3. Sodium azide is primarily used as a precursor in the synthesis of other chemical compounds and materials.
Automatically generated - may contain errors
Lab products found in correlation
Fv1000 ix81, by Olympus (3 mentions)
Ethylenediaminetetraacetic acid, by Merck Group (2 mentions)
Quercetin, by Merck Group (2 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Sodium acetate trihydrate, by Merck Group (1 mentions)
Agar powder, by Merck Group (1 mentions)
Sodium taurodeoxycholate hydrate, by Merck Group (1 mentions)
2 4 6 tri 2 pyridyl 1 3 5 triazine, by Merck Group (1 mentions)
K2s2o8, by Merck Group (1 mentions)
Mgso4, by Junsei (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
2 4 6 tripyridyl s triazine, by Merck Group (1 mentions)
Xylene, by Merck Group (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Fecl3 6h2o, by Merck Group (1 mentions)
Chloroform, by Avantor (1 mentions)
Tris hcl buffer, by Merck Group (1 mentions)
Pyrogallol, by Merck Group (1 mentions)
Na2co3, by Daejung Chemicals (1 mentions)
7 protocols using sodium azide
1
Visualizing DAF-16 Localization in C. elegans
2
Measuring intestinal lipofuscin autofluorescence
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The autofluorescence of intestinal lipofuscin was measured as an index of senescence between days 10 and 18 of adulthood. Randomly selected worms from the plate lawned with E. coli OP50 or Weissella species were washed three times with M9 buffer. Worms were then placed onto a 5% agar pad coated with 10 mM sodium azide (Junsei Chemical, Tokyo, Japan) in M9 buffer to induce anaesthesia. Lipofuscin autofluorescence images were taken using blue excitation light (405− 488 nm), a DAPI (4′,6-diamidino-2-phenylindole) channel of laser confocal scanning microscope (Olympus Ix81-FV1000, Japan)51 (link). Fluorescence was quantified on days 14, 16 and 18 using ImageJ software (National Institutes of Health, Bethesda, MD, USA) to determine the lipofuscin levels. Three independent experiments were performed with over 30 worms for each bacterial species on each day.
3
Comprehensive Microbiological Evaluation Protocol
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Sodium azide and MgSO4 were obtained from Junsei (Japan). Oxgall, MRS agar, MRS broth, tryptone soy agar and broth, and nutrient agar and broth were purchased from Difco (USA). HCl and Na2CO3 were obtained from Daejung (Korea). Agar powder, β-mercapto ethanol, sodium dodecyl sulfate (SDS), σ-nitrophenyl-β-D-galactopyranoside (ONPG), trichlo acetic acid, 2,2-diphenyl-1-picrylhydrazyl, 2, 4, 6-tripyridyl-s-triazine (DPPH), iron (III) chloride hexahydrate 97% A.C.S reagent, 2,4,6-tri(2-pyridyl)-1,3,5-triazine (TPTZ), FeCl3•6H2O, Pyrogallol, K2S2O8, ethylene diamine tetra acetic acid (EDTA), acetate buffer (pH 3.6), Tris-HCl buffer, xylene, sodium acetate trihydrate, and sodium taurodeoxycholate hydrate were obtained from Sigma (USA). A gram stain kit was obtained from YD Diagnostics (Korea). An API 50 CHL kit was obtained from Bio Merieux Ltd. (France). 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid was obtained from Fluka (Germany). Lactic acid was obtained from Konto (Japan). BCP agar was obtained from Aiken (Japan). Skim milk was obtained from Seoul Dairy Co-op. (Korea). Chloroform was obtained from JT Baker (USA). NaOH pellets were obtained from Mallinckrodt (USA). Finally, the sterilized 0.45 μm filters were obtained from Millipore (Ireland).
4
Ginsenoside Compound Characterization
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DL-glyceraldehyde dimer, adenine dinucleotide phosphate (NADPH), β-nicotinamide bovine serum albumin (BSA), and quercetin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Human recombinant AR (0.4 units) was purchased from Wako Chemicals (Osaka, Japan). Sodium azide was purchased from Junsei Chemical Co. (Tokyo, Japan). Ginsenoside Rb2 (>95%), ginsenoside Rb3 (>95%), protopanaxadiol (purity >85%), ginsenoside Rf (>95%), ginsenoside Rc (>98%), protopanaxatriol (>96%), ginsenoside Re (>97%), (20S) ginsenoside Rg3 (>98%), ginsenoside Rd (>95%), ginsenoside Rg1(>90%), ginsenoside Rh2 (>97%), compound K (>96%), sodium hydroxide, ginsenoside Rb1 (>98%), quercetin (>95%), ginsenoside Rh1(>90%), glucose, dimethyl sulfoxide (DMSO), and sorbitol were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). (20R) ginsenoside Rg3 (purity > 96%), (20S) ginsenoside Rg2 (>90%), ginsenoside Ra1 (>96%), ginsenoside Ra2 (>98%), ginsenoside Rs1 (>97%), ginsenoside Rs2 (>90%), (20R) ginsenoside Rg2 (>99%) were purchased from Selleckchem Co. (Cedarlane, ON, Canada). Unless otherwise noted, all additional chemicals and solvents were reagent grade and purchased from Merck (Darmstadt, Germania), Fluka (Buchs, Switzerland), Duksan Pure Chemical Co. (Ansan, South Korea), or Sigma-Aldrich Co.
5
Lipofuscin Autofluorescence in C. elegans
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The autofluorescence of lipofuscin in 14-day-old adult C. elegans was measured as an aging index. Randomly selected worms from each group were placed onto 5% agar pads coated with 10 mM sodium azide (Junsei Chemical, Tokyo, Japan) in M9 buffer for anesthetization. Images of lipofuscin autofluorescence at a blue excitation wavelength (405–488 nm), which captures 4',6-diamidino-2-phenylindole (DAPI), were acquired with a laser confocal scanning microscope (Olympus Ix81-FV1000)40 (link). Fluorescence was quantified using FV10-ASW1.1 software (Olympus) to measure lipofuscin accumulation. Three independent experiments were conducted, and 10 worms were included in each group for each measurement.
6
Inhibitory Effects of Natural Compounds on PTP1B and α-Glucosidase
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Protein tyrosine phosphatase 1B (PTP1B; human recombinant) was purchased from Biomol International LP (Plymouth Meeting, PA), dithiothreitol (DTT) was purchased from Bio‐Rad Laboratories (Hercules, CA), and sodium azide was purchased from Junsei Chemical Co. (Tokyo, Japan). Yeast α‐glucosidase, p‐nitrophenyl phosphate (pNPP), p‐nitrophenyl α‐D‐glucopyranoside (pNPG), ethylenediaminetetraacetic acid (EDTA), β‐nicotinamide adenine dinucleotide phosphate (NADPH), Folin–Ciocalteu reagent, gallic acid, Trolox, ascorbic acid, ursolic acid, acarbose, quercetin, DPPH, l‐penicillamine, dl‐glyceraldehyde, bovine serum albumin (BSA), sodium bicarbonate, dimethyl sulfoxide (DMSO), D‐(−)‐fructose, D‐(+)‐glucose, aminoguanidine, and rosiglitazone were purchased from Sigma‐Aldrich (St. Louis, MO). Fetal bovine serum (FBS), minimum essential medium (MEM), sodium pyruvate, penicillin–streptomycin, nonessential amino acids, and the fluorescent D‐glucose analog and glucose tracer 2‐[N‐(7‐nitrobenz‐2‐oxa‐1, 3‐diazol‐4‐yl) amino]‐2‐deoxy‐D‐glucose (2‐NBDG) were purchased from Life Technologies (Carlsbad, CA). Human insulin was purchased from Eli Lilly (Fegersheim, France). All other chemicals and solvents used were purchased from E. Merck, Fluka, and Sigma‐Aldrich.
7
Quantifying Intestinal Lipofuscin Autofluorescence
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The autofluorescence of intestinal lipofuscin was measured as an index of senescence at day 18 of adulthood. Day-18 worms from the RNAi-bacteria NGM plates were picked and mounted onto a 2% agarose pad coated with 10 mM sodium azide (Junsei Chemical, Tokyo, Japan) in M9 buffer to induce anaesthesia. Lipofuscin autofluorescence images were captured using a DAPI (4′,6-diamidino-2-phenylindole) channel from an AxioCam HRc CCD digital camera (Zeiss Corporation) with a Zeiss Axio Scope A1 compound microscope (Zeiss Corporation). The fluorescence intensity of the animals was quantified by using ImageJ software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, MD, USA, http://rsb.info.nih.gov/ij/ ).
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