Freshly dissected tibias from 8 to 12-week-old mice were fixed for 6–8 h in 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4 °C. Tibias were sectioned perpendicular to the long axis into 300-μm thick sections using a Leica VT100 S vibratome. Sections were blocked in a staining solution containing anti-CD16/32 mouse Fc-blocking antibody overnight on a rotator at room temperature. The staining solution contained 10% dimethyl sulfoxide (Sigma, #276855), 0.5% IgePal630 (Sigma, # I3021), and 5% donkey serum (Jackson Immuno, # 017-000-121) in PBS. Bone sections were stained with green fluorescent protein (GFP), c-Kit, and laminin antibodies for 3 days, washed overnight in several changes of PBS then incubated for 3 days in a staining solution containing secondary antibodies. Antibody dilutions are listed in Supplementary Table. 7 . The stained bone sections were dehydrated in a methanol series and cleared in benzyl alcohol: benzyl benzoate 1:2 mix (BABB clearing). 3D microscopy of the bone marrow was performed on a Confocal/Multiphoton Zeiss Leica SP8 resonant scanning confocal microscope with two-photon excitation. Confocal tiled Z-stack images were rendered in 3D and analyzed using Imaris v. 7.7.1 software.
Igepal630
Manufactured by Merck Group
IgePal630 is a nonionic detergent used in biochemical applications. It is a polyoxyethylene octylphenyl ether that functions as a solubilizing agent and emulsifier.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (6 mentions)
Donkey serum, by Jackson ImmunoResearch (3 mentions)
Prolong gold antifade, by Thermo Fisher Scientific (2 mentions)
Streptavidin af647, by Thermo Fisher Scientific (2 mentions)
Photosensitive film, by Thomas Scientific (2 mentions)
Hrp linked anti mouse igg, by Cell Signaling Technology (2 mentions)
Mouse ha 11 clone 16b12, by BioLegend (2 mentions)
Anti mouse igg ap linked, by Promega (2 mentions)
Hyblot, by Thomas Scientific (2 mentions)
Nbt bcip substrate, by Promega (2 mentions)
Complete mini protease inhibitor cocktail, by Roche (2 mentions)
Bradford assay kit, by Bio-Rad (2 mentions)
Anti tdtomato, by Takara Bio (2 mentions)
Alexa fluor 647 affinipure f ab 2 fragment donkey anti chicken igy, by Jackson ImmunoResearch (2 mentions)
Alexa fluor 488 affinipure f ab 2 fragment donkey anti rabbit igg, by Jackson ImmunoResearch (2 mentions)
555 conjugated donkey anti goat antibody, by Thermo Fisher Scientific (2 mentions)
Sp8 confocal microscope, by Leica (2 mentions)
Chicken anti gfp, by Aves Labs (2 mentions)
Cutsmart buffer, by New England Biolabs (2 mentions)
Nb bbvci, by New England Biolabs (2 mentions)
12 protocols using igepal630
1
3D Imaging of Murine Bone Marrow
2
Isolation and Analysis of α-catulin-GFP+ Cells
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Bone marrow cells from a-catulinGFP/+ mice were prepared for flow cytometric analysis as described above. Biotinylated anti-c-kit antibody (2B8 clone, 13-1171-85, eBiosciences) followed by streptavidin-AF647 (S32357, Life Technologies) were used to stain bone marrow cells. α-catulin-GFP+c-kit+ cells were sorted into a drop of staining medium on a slide coated with poly-D-lysine (0.5mg/ml poly-D-lysine in water was used to coat the slides over night at room temperature). Slides were incubated for 45 minutes at 4°C to let the sorted cells attach to the slide surface. Then a 16% PFA stock solution was added gently into the drop of staining medium to achieve a final PFA concentration around 4%. Cells were fixed for 10 minutes at room temperature and washed multiple times with PBS. Then the cells were stained with DAPI (2 μg/ml in PBS), with 0.1% IgePal630 (Sigma) for 30 minutes, followed by multiple washes in PBS. Prolong gold antifade (Life Technologies) was used to mount the cells. An LSM780 confocal microscope (Zeiss) was used to image the cells and Imaris software was used to measure cell diameter.
3
Isolation and Analysis of α-catulin-GFP+ Cells
4
Perovskite Nanocrystal Synthesis and Characterization
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Cesium carbonate (Cs2CO3, 99 %), Lead(II) bromide (PbBr2, ≥98 %), lead(II) iodide (PbI2, 99.999 %), lead(II) Chloride (PbCl2, 98 %), 1-octadecene (ODE, 90 %), oleic acid (OA, 95 %), oleylamine (OAm), hexane anhydrous (95 %), toluene anhydrous (99.8 %), isobornyl acrylate, dipentaerythritol penta-/hexa-acrylate, diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, IGEPAL 520, IGEPAL 630, diethylene glycol (99 %), triethylene glycol (99 %), glycerol (≥99 %), propylene glycol monoether acetate (PGMEA, ≥9.5), and molybdenum trioxide (MoO3, 99.97 %) from Sigma-Aldrich were used without additional purification. N,N-bis(naphthalen-1-yl)-N,Nbis(phenyl)benzidine (NPB), tris(4-carbazoyl-9-ylphenyl)amine (TCTA), N,N′-dicarbazolyl-3,5-benzene (mCP), bis[2-(4,6-difluoro -phenyl)pyridinato-C2,N](picolinato)iridium(III) (FIrpic), 2,2,2′′- (1,3,5-benzinetriyl)-tris(1-phenyl-1-H-benzimidazole) (TPBI), and lithium fluoride (LiF) (1 nm) were purchased from OSM. Aluminum (Al) and silver (Ag) were obtained from TASCO. Indium-tin oxide (ITO) was purchased from GUNZE [6] , [21] .
5
Caspase Activity Assay in Cell Lysates
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Cells were harvested, pelleted by centrifugation at 350 g, washed and resuspended in 50 μl of PBS. Twenty-five μl was transferred to duplicate wells of a microtiter plate and snap-frozen in liquid nitrogen. To initiate the reaction, 50 μM of the caspase substrate Ac-Asp-Glu-Val -Asp-α -(4-methyl-coumaryl-7-amide) (Peptide Institute Inc., 3171-v) in assay buffer (100 mM HEPES, pH 7.5, 10% sucrose [Sigma-Aldrich, S7903], 0.1% CHAPS [Sigma-Aldrich, C9426], 5 mM DTT [Sigma-Aldrich, D0632] and 0.0001% Igepal-630 [Sigma-Aldrich, I8896], pH 7.25) was added to cell lysates. Liberated free AMC was measured by a Wallac Victor 1420 Multilabel counter (Perkin Elmer Life Sciences, Bioscience research building, National University of Ireland Galway) using 355 nm excitation and 460 nm emission wavelengths at 37°C at 60 s intervals for 25 cycles. The data were analyzed by linear regression and enzyme activity was expressed as nM of AMC released × min−1 × mg−1 total cellular protein.
6
Spectrophotometric Assay of G6PD Activity
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G6PD activity was determined by spectrophotometrically monitoring the increase of absorbance at 340 nm (A340) due to the reduction of NADP+ in the presence of glucose-6-phosphate. Briefly, cells were scraped and resuspended in ice-cold PBS containing 0.3% (v/v) Igepal 630 (Sigma-Aldrich) and 1X Complete protease inhibitor cocktail. Cell suspensions were sonicated three times for 30 s with 1 min rest intervals in a Bioruptor device (Diagenode) at 4ºC. The resulting lysates were cleared by centrifugation at 10,000 g at 4ºC for 10 min, and supernatants used for the assays. The protein content was measured with the Pierce BCA protein assay kit (ThermoFisher Scientific). G6PD reaction mixtures consisted of 30 mM Tris–HCl, pH 7.5, 6 mM MgCl2, 0.5 mM NADP + , 1 mM glucose-6-phosphate, and different amounts of cellular extracts in a final volume of 1 ml. Reactions were initiated by adding the cell extract to the reaction cuvette and analysed by recording A340 continuously in a UV 1800 spectrophotometer (Shimadzu) for 5 min at room temperature. Enzyme activity was calculated from the A340 slopes and expressed as nanomole of NADPH produced per minute and milligram of protein.
7
Western Blot Protein Analysis
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Cells were suspended and then sonicated in RIPA buffer (150 mM NaCl, 50 mM Tris (pH 8.0), 1% Igepal-630 (Sigma-Aldrich), 0.5% sodium deoxycholate, 1 tablet Complete Mini protease inhibitor cocktail (Roche, Basel, Switzerland)) and mixed end-over-end for 1 h at 4 °C. Lysates were cleared by centrifugation, and the total protein was quantified with a Bradford assay kit according to the manufacturer’s instructions (BioRad, Hercules, CA). Equal amounts of protein were mixed with Laemmli buffer and loaded onto 12% gels and separated by SDS-PAGE. Proteins then were transferred to PVDF membranes using a semi-dry method. Membranes were blocked with PBSTween (PBS with 0.1% Tween-20 (v/v)) containing 5% dry milk (w/v), incubated in primary antibody (either 1:2,000 mouse HA.11 clone 16B12 (BioLegend, San Diego, CA, #MMS-101R) or 1:6,000 mouse anti-β tubulin (Hybridoma Bank, Iowa City, IA, #E7)), washed with PBSTween, incubated in secondary antibody (1:6,000 anti-mouse IgG AP-linked (Promega, Madison, WI) or anti-mouse IgG HRP-linked (Cell Signaling Technology, Beverly, MA)), and washed again with PBSTween. Antibody binding was detected using either BCIP/NBT substrate (Promega) or ECL substrate (HyBlot, Denville Scientific, Hollison, MA). For ECL detection, membranes were exposed to photosensitive film (Denville Scientific). All antibodies were diluted in PBSTween containing 5% dry milk.
8
Western Blot Protein Analysis
9
Whole-mount Imaging of Bone Samples
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Femurs were longitudinally cut in half, then stained, and deep imaged as described previously21 (link). The staining solution contained 10% DMSO, 0.5% IgePal630 (Sigma), and 5% donkey serum (Jackson Immuno) in PBS. Half bones were stained for 3 days at room temperature with primary antibodies. Then specimens were washed 3 times in PBS at room temperature for one day and put into staining solution containing secondary antibodies for 3 days followed by a one-day wash. Antibodies used for whole mount staining included chicken anti-GFP (Aves Labs, 1:200), anti-tdTomato (Takara, 1:200), Alexa Fluor 647-AffiniPure F(ab′)2 Fragment Donkey Anti-Chicken IgY (1:250), Alexa Fluor 488-AffiniPure F(ab′)2 Fragment Donkey Anti-Rabbit IgG (1:250), Alexa Fluor 488-AffiniPure F(ab′)2 Fragment Donkey Anti-Rabbit IgG (all from Jackson ImmunoResearch, 1:250), and 555--conjugated donkey anti-goat antibody (Invitrogen, 1:250). Images were acquired using a Leica SP8 confocal microscope. Images were rendered in 3D and analyzed using Bitplane Imaris v7.7.1.
10
Singly Nicked Plasmid Preparation and Ligation
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