OI-iPSC constructs were fixed with 10% neutral-buffered formalin solution (Wako Pure Chemical) for 1 day. Next, the specimens were embedded in paraffin, followed by the preparation of sequential sections with 4 μm thickness using a microtome. Subsequently, the paraffin-embedded sections were evaluated using histological and immunofluorescent staining. The histological staining performed in this study was standard hematoxylin and eosin (HE) staining, methylene blue-counterstained von Kossa staining, and Movat's pentachrome staining. The paraffin-embedded sections were deparaffinized with xylene (Wako Pure Chemical) and hydrated through graded alcohol to distilled water prior to HE staining and methylene blue-counterstained von Kossa staining. For methylene blue-counterstained von Kossa staining, the specimens were incubated with 5% silver nitrate (AgNO3; Wako Pure Chemical) under ultraviolet (UV) light for 10 minutes, then rinsed in two changes of distilled water. Next, the slides were incubated in 5% sodium thiosulfate (Sigma-Aldrich) for 5 minutes to eliminate unreacted silver and washed with running water for 5 minutes. Subsequently, nuclear counterstaining was performed using 1% methylene blue (Wako Pure Chemical) in 10 mM borate buffer. For Movat's pentachrome staining, the staining was performed as previously reported [24 (link)–26 (link)].
Methylene blue
Manufactured by Fujifilm
Sourced in Japan
Methylene blue is a lab equipment product used as a stain or dye in various applications. It is a blue crystalline solid that is soluble in water. Methylene blue is commonly used in microscopy, biochemistry, and analytical chemistry for its staining properties.
Automatically generated - may contain errors
Lab products found in correlation
Ethanol, by Fujifilm (3 mentions)
Sodium hydroxide (naoh), by Fujifilm (2 mentions)
Agarose, by BD (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Diethylaminoethyl dextran, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
Trypsin, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Fujifilm (1 mentions)
2 7 dichlorodihydrofluorescein diacetate dcfh da, by Cayman Chemical (1 mentions)
Copper 2 chloride dihydrate, by Fujifilm (1 mentions)
Hoechst 33342 solution, by Fujifilm (1 mentions)
Calcein am, by Thermo Fisher Scientific (1 mentions)
Lysotracker green dnd 26, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Bh 2 microscope, by Olympus (1 mentions)
Sodium chloride (nacl), by Fujifilm (1 mentions)
Hydrochloric acid (hcl), by Fujifilm (1 mentions)
Styrene, by Fujifilm (1 mentions)
16 protocols using methylene blue
1
Histological Evaluation of OI-iPSC Constructs
2
Colony-Formation Assay for Radiation Sensitivity
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The colony-formation assay was performed as described previously [17 (link), 18 (link)] Colonies were fixed and stained with methylene blue solution (0.25% methylene blue (FUJIFILM Wako) in 90% ethanol (FUJIFILM Wako). The number of surviving colonies that included 50 cells or more was counted. The surviving fraction was calculated based on the plating efficiency [19 (link)]. The sensitivity enhancement ratio at 6 Gy (SER) was calculated using the equation SER(6) = log Sf(MHY1485, 6) / log Sf(DMSO, 6), where Sf(MHY1485, 6) and Sf(DMSO, 6) are the surviving fractions at 6 Gy following exposure to MHY1485 and DMSO, respectively [20 (link)].
3
Subcutaneous Hamster Skin Cancer Xenograft Model
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Example 2
A Syrian hamster (6-week-old male) was subcutaneously transplanted with 16,500,000 RPMI 1846 skin cancer cells derived from the same species to obtain a subcutaneously-transplanted hamster model as described below. The distal end of the permeation apparatus 2 was inserted into a tumor tissue (target) as shown in
As shown in
4
Plaque Assay Protocol for MDCK Cells
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Plaque assays were performed as described previously10 (link), 11 (link). Briefly, Madin-Darby canine kidney (MDCK) cells (Lonza, Walkersville, MD, USA) were maintained at 37 °C in a humidified 5% CO2 chamber under stationary conditions. Each well of a 6-well plate was seeded with 1 × 106 cells and cultured in α-minimum essential medium (MEM; GIBCO/Invitrogen, Carlsbad, CA, USA) containing 10% foetal bovine serum (FBS), 100 units/mL penicillin (GIBCO), and 100 μg/mL streptomycin (GIBCO). After two washes with serum-free Dulbecco’s modified Eagle’s medium (DMEM; GIBCO/Invitrogen), the cells were maintained in serum-free DMEM at 37 °C for 1 h. Then, each well was overlaid with 200 μL of diluted BAL (10−3, 10−4, and 10−5 dilutions) and incubated at 37 °C for 1 h. After one wash in serum-free DMEM, the cells were overlaid with serum-free DMEM containing 0.8% agarose (Becton, Dickinson and Company, Sparks, MD, USA), 0.1% diethylaminoethyl-Dextran (Sigma-Aldrich), and 7 μg/mL trypsin (Sigma-Aldrich). The cells were cultured at 37 °C for 72 h, fixed in 10% formaldehyde (Wako Pure Chemical Industries, Ltd., Osaka, Japan), and then stained with 0.037% methylene blue (Wako Pure Chemical Industries, Ltd.). Each experiment was performed in duplicate.
5
Multimodal Cellular Imaging Protocol
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Copper(II) chloride dihydrate
(CuCl2·2H2O), gadolinium(III) chloride
hexahydrate (GdCl3·6H2O), sodium hydroxide
(NaOH), bovine serum albumin, sodium sulfide pentahydrate (Na2S·5H2O), methylene blue, and Hoechst 33342
solution were purchased from Fujifilm Wako Pure Chemical Co. (Osaka,
Japan). Sulfo-NHS-Cy5.5 ester was obtained from Abcam (Cambridge,
UK). Cyclo(RGDfk) and 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA)
were obtained from Cayman Chemical (Michigan, USA). Sulfo-NHS-acetate
was purchased from BroadPharm, California (USA). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC), N-hydroxysulfosuccinimide (sulfo-NHS),
Alexa Fluor 488 phalloidin, LysoTracker Green DND-26, calcein AM,
and propidium iodide (PI) were obtained from Thermo Fisher Scientific
(Massachusetts, USA). All other chemicals of analytical reagent grade
were obtained from qualified reagent suppliers.
(CuCl2·2H2O), gadolinium(III) chloride
hexahydrate (GdCl3·6H2O), sodium hydroxide
(NaOH), bovine serum albumin, sodium sulfide pentahydrate (Na2S·5H2O), methylene blue, and Hoechst 33342
solution were purchased from Fujifilm Wako Pure Chemical Co. (Osaka,
Japan). Sulfo-NHS-Cy5.5 ester was obtained from Abcam (Cambridge,
UK). Cyclo(RGDfk) and 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA)
were obtained from Cayman Chemical (Michigan, USA). Sulfo-NHS-acetate
was purchased from BroadPharm, California (USA). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC), N-hydroxysulfosuccinimide (sulfo-NHS),
Alexa Fluor 488 phalloidin, LysoTracker Green DND-26, calcein AM,
and propidium iodide (PI) were obtained from Thermo Fisher Scientific
(Massachusetts, USA). All other chemicals of analytical reagent grade
were obtained from qualified reagent suppliers.
6
Enumeration of Aberrant Crypt Foci
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The frequency of ACF was determined as previously described (10 (link),23 (link)). The colon samples were fixed with 10% buffered formalin, stained with methylene blue (0.5% in distilled water; Wako Pure Chemical Industries, Ltd.) for 20 sec and then placed on microscope slides to count the number of ACF using a BH2 Olympus microscope (Olympus, Tokyo, Japan). The number of ACF was recorded along with the number of aberrant crypts (ACs) in each focus. Data are presented as per unit area (cm2).
7
Anionic Surfactant Extraction Efficiency
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Sodium dodecyl sulfate (SDS: CH3[CH2]11OSO3Na, 99.0%) was purchased from Wako Pure Chemicals (Tokyo, Japan) and used as the standard anionic surfactant. Solvents to compare the AS extraction efficiency in this study were chloroform (CHCl3, 99.8%), methyl isobutyl ketone (MIBK: CH3COC4H9, 99.0%), 1,2-dichloroethane (DCE: ClC2H4Cl, 99.8%), dichloromethane (CH2Cl2, 99.9%), benzene (C6H6, 99.5%), and n-hexane (C6H14, 96.0%). Methylene blue (MB: C16H18N3SCl·3H2O, 97.0%) and the above solvents were purchased from Wako Pure Chemicals (Tokyo, Japan), except for DCE and MIBK, which were obtained from Junsei Chemical (Tokyo, Japan) and Daejung Chemical (Siheung, Korea), respectively. Concentration of MB as a cationic dye was prepared at 0.025% solution. All chemicals used were reagent grade.
8
Versatile Polymer Synthesis Protocols
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Titanium tetraisopropoxide (TTIP, 95%), methylamine aqueous solution (40%), acetonitrile (99.5%), ethanol (99.5%), styrene (St, 99%), p-styrenesulfonic acid sodium salt (NaSS, 80%), potassium persulfate (KPS, 95%), sodium chloride (99.5%), tetraethyl orthosilicate (TEOS, 95%), hydrochloric acid (HCl, 0.10 mol/L), and methylene blue (MB, 98.5%) were purchased from FUJIFILM Wako Pure Chemical Corp. (Osaka, Japan). 3-Methacryloxypropyltrimethoxysilane (MPTMS, 95%) was obtained from Shin-Etsu Chemical Co. (Tokyo, Japan). The inhibitor for the St monomer was removed using an inhibitor removal column. Deionized water (>18.2 MΩ cm) was prepared for experimental use.
9
Colorimetric Analysis Protocol
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Phosphate buffer powder (consisting of Na2HPO4 and KH2PO4), methylene blue, sodium hydroxide (NaOH), potassium iodide (KI), and ammonium molybdate tetrahydrate ((NH4)6Mo7O24·4H2O) were obtained from FUJIFILM Wako Pure Chemicals Corporation (Osaka, Japan). Potassium hydrogen phthalate (C6H4(COOK)(COOH)) was purchased from Tokyo Chemical Industry Co, Ltd (Tokyo, Japan). These chemicals were used without further purification.
10
Organic Synthesis with PPTS Catalyst
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LA, pyridinium p-toluenesulfonate (PPTS), and chloroform-d (CDCl3) were obtained from Sigma (St. Louis, MO, USA). Methylene blue, 2-methoxypropene (MxP), N,N’-dicyclohexylcarbodiimide (DCC), 4-(dimethylamino)pyridine (DMAP), and dimethyl sulfone were purchased from Wako Pure Chemical Co. (Osaka, Japan). All other reagents were of the highest grade available.
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