The frozen sections were fixed with 4% paraformaldehyde (Sigma #P6148) and permeabilized with 0.25% Triton X-100 (Fisher Scientific #BP151–100). After a wash with PBS/Tween-20 (Fisher Scientific #BP337–100), slides were treated with 3 M hydrochloric acid (Fisher Scientific #A144S-500) for 10 min to open the nucleus structure if needed. Slides were blocked with 10% donkey serum (Sigma #D9663) in 2% BSA (Sigma) in PBS/Tw-20 for 1 h at room temperature. Afterwards, slides were probed with primary antibodies (CD31 1:25, BD #550274; BrdU-APC 1:50, BD; Alexa Fluor 488 Mouse anti-BrdU 1:10, BD #558599; Flag M2 1:500, Sigma #F1804) and incubated overnight at 4 °C. The next day, slides were washed and incubated with the fluorescence-conjugated secondary antibody (AF488 donkey anti-rat 1:300, Invitrogen #A-21208; AF594 donkey anti-mouse 1:300, Invitrogen #A-21203; AF594 donkey anti-rat 1:300, Invitrogen #A-21209). Images were taken with a confocal microscope LSM880 (Zeiss) and analyzed by Zen software (Zeiss).
Bp337 100
Manufactured by Thermo Fisher Scientific
The BP337-100 is a general-purpose laboratory balance from Thermo Fisher Scientific. It is designed to accurately measure the weight of samples up to 100 grams. The balance features a digital display and provides precise measurements in different units of mass.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880, by Zeiss (4 mentions)
Bp151 100, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
A9418, by Merck Group (2 mentions)
A21208, by Thermo Fisher Scientific (1 mentions)
A21203, by Thermo Fisher Scientific (1 mentions)
A21209, by Thermo Fisher Scientific (1 mentions)
D9663, by Merck Group (1 mentions)
P6148, by Merck Group (1 mentions)
Zen software, by Zeiss (1 mentions)
Flag m2, by Merck Group (1 mentions)
X100 100ml, by Merck Group (1 mentions)
Goat anti mouse irdye 680, by LI COR (1 mentions)
Goat anti rabbit irdye 800, by LI COR (1 mentions)
Ab142227, by Abcam (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Bp2818500, by Thermo Fisher Scientific (1 mentions)
Res20400 a702x, by Merck Group (1 mentions)
Tmb elisa substrate, by Thermo Fisher Scientific (1 mentions)
Bp1600 100, by Thermo Fisher Scientific (1 mentions)
9 protocols using bp337 100
1
Immunofluorescence Staining of Frozen Tissue
2
Immunofluorescence Staining Protocol
3
Western Blot Analysis of Protein Targets
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Cells were lysed for 20 min on ice in lysis buffer containing 50 mM Tris HCl (pH 7.6), 125 mM NaCl, 5% glycerol, 0.2% NP-40 (ab142227, Abcam), 1.5 mM MgCl2, and protease and phosphatase inhibitor cocktail (78441, Thermo Scientific). Cell lysates were analyzed using SDS–PAGE and 10% polyacrylamide gels and transferred to a Whatman Protran nitrocellulose membrane (1620115, Biorad). Membranes were blocked for 1 h at room temperature in Odyssey blocking buffer (92740000, LI-COR). After blocking, membranes were incubated at 4°C overnight with primary antibody in Odyssey blocking buffer. Subsequently, membranes were washed three times for 10 min in PBS containing 0.1% Tween 20 (BP337-100, Fisher Scientific) and incubated for 1 h at room temperature with secondary antibody in Odyssey blocking buffer. Finally, membranes were washed three times for 10 min in PBS/Tween 20 and rinsed in Milli-Q-H2O. The Odyssey Classic infrared imaging device (LI-COR) was used for signal detection. The following primary antibodies were used: mouse anti-GAPDH (MA5-15738, Thermofisher, 1/3000 dilution) and rabbit anti-zyxin (ABD1463, Millipore, 1/1000 dilution), which was a kind gift of Mary Beckerle’s laboratory (University of Utah, Salt Lake City, UT). The following LI-COR secondary antibodies were used at a 1/10,000 dilution: goat anti-rabbit IRdye800 (925-32211), goat anti-mouse IRdye680 (925-68070).
4
Optimized Al(III) and Fe(III) Hematoxylin Staining
5
Quantitative ELISA for Human Immunoglobulins
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Goat anti-human kappa cross-absorbed against mouse IgG (Southern Biotech catalog # 2061-01) and goat anti-human lambda cross-absorbed against mouse IgG (Southern Biotech catalog # 2071-01) were coated onto 96-well Nunc Maxisorp flat-bottomed plates at 2 μg/mL in coating buffer (0.1 M sodium carbonate, 0.1 M sodium bicarbonate, 0.02% NaN3, pH 9.6) overnight at 4°C. Coating buffers were aspirated, and wells were blocked with 2% BSA (blocking buffer) (Fisher Bioreagents catalog # BP1600-100), for 1 h at 37°C. Heat-inactivated serum samples were diluted in blocking buffer in a separate polypropylene plate. The plates then were washed 4 × with 1 × PBS + 0.05% Tween-20 (PBST) (Fisher Bioreagents catalog # BP337-100), followed by addition of 50 μL of respective serum dilutions and was incubated for 1 h at 4°C. The ELISA plates were again washed 4 × in PBST, followed by addition of 50 μL of 1:2,000 Goat Anti-Human IgG Fc, Multi-Species (Southern Biotech catalog # 2014-05). Plates were incubated for 1 h at 4°C. Plates were washed with 4 × PBST, followed by 100 μL of TMB-ELISA substrate (Thermo Fisher Scientific catalog # 34028) and incubated at room temperature for 3 to 5 min. Color development was observed and reactions were stopped with 50 μL of 2N sulfuric acid. Optical density (450 nm) measurements were determined using a microplate reader (Bio-Rad).
6
Western Blot Analysis of Protein Expression
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CFBE or 16HBE cells were washed twice with cold PBS and lysed with sample buffer containing 31.25 mM Tris HCl (30721; Sigma-Aldrich), pH 6.8; 1.5% (vol/vol) sodium dodecyl sulphate (SDS) (15553; Gibco); 10% (vol/vol) glycerol (92025; Sigma-Aldrich); 50 mM dithiothreitol (DTT) (D0632; Sigma-Aldrich), and protease inhibitor cocktail (11697498001; Roche). Benzonase (E1014; Sigma-Aldrich) 25 U/ml was also added to shear the DNA. 25–30 μg of protein were loaded onto polyacrylamide gels (4% for stacking and 7/10% for resolving gels) to perform SDS/PAGE. Transfer onto polyvinylidene difluoride (PVDF) membranes (IPVH00010; Merck Millipore) was performed using a wet-transfer system. The membranes were blocked for 1 h with 5% (wt/vol) non-fat milk (NFM) in PBS supplemented with Tween 20 (BP337-100; Fisher BioReagents). This was followed by incubation with the primary antibody overnight at 4°C, with gentle shaking. HRP-conjugated secondary antibodies were applied for 1 h at RT. All the antibodies were diluted in the blocking solution. Membrane luminescence was detected on a Chemidoc XRS+ system (170-8265; Bio-Rad). Quantification of band intensity was performed using the Image Lab software (170-9690; Bio-Rad), which integrates peak area. All measurements were normalized against loading controls (calnexin or GAPDH). A list of primary antibodies can be found in Table S1.
7
Optimizing Formamide Concentration for RNA Detection
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Following (Raj et al., 2006 (link)), a range of
formamide concentrations was initially tested to empirically determine the optimal
value. 20% (w/v) formamide gave the best results in that it was high enough so
that background noise due to non-specific binding was low, while still low enough
so that the fluorescence signal from target mRNA molecules was not impaired.
10 ml of wash solution contains 1.76 ml of formamide (Ambion, AM9342), 1 ml of 20×
SSC (Ambion, AM9763), and 10 μl Tween-20 (Fisher Scientific, BP337-100). Wash
solution was made fresh and stored on ice until use. 10 ml of hybridization
solution contains 1 g of dextran sulfate (Sigma, D8906), 1.76 ml of formamide, 10
mg of E. coli tRNA (Sigma, R4251), 1 ml of 20× SSC, 40 μl of 50
mg/ml BSA (Ambion, AM2616), and 100 μl of 200 mM ribonucleoside vanadyl complex
(New England Biolabs, Ipswich, NY, S1402S). Hybridization solution was filter
sterilized and aliquots of 500 μl were stored at -20°C.
formamide concentrations was initially tested to empirically determine the optimal
value. 20% (w/v) formamide gave the best results in that it was high enough so
that background noise due to non-specific binding was low, while still low enough
so that the fluorescence signal from target mRNA molecules was not impaired.
10 ml of wash solution contains 1.76 ml of formamide (Ambion, AM9342), 1 ml of 20×
SSC (Ambion, AM9763), and 10 μl Tween-20 (Fisher Scientific, BP337-100). Wash
solution was made fresh and stored on ice until use. 10 ml of hybridization
solution contains 1 g of dextran sulfate (Sigma, D8906), 1.76 ml of formamide, 10
mg of E. coli tRNA (Sigma, R4251), 1 ml of 20× SSC, 40 μl of 50
mg/ml BSA (Ambion, AM2616), and 100 μl of 200 mM ribonucleoside vanadyl complex
(New England Biolabs, Ipswich, NY, S1402S). Hybridization solution was filter
sterilized and aliquots of 500 μl were stored at -20°C.
8
Carnegie Stage 6 Marmoset Cryosection Protocol
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Carnegie stage 6 marmoset cryosections originated from embryos of a recently published52 (link) study. Briefly, slides were thawed at room temperature and fixed for 8 min in 4% PFA/PBS solution (15714S, Electron Microscopy Sciences/Thermo Fisher Scientific), and then washed three times with PBS. Permeabilization was performed in 0.25% Triton X-100 (13444259, Thermo Fisher Scientific) in 0.3% polyvinyl pyrrolidone/PBS (Thermo Fisher Scientific) for 30 min at room temperature. The slides were rinsed 3 times with PBS and incubated for 30 min in blocking buffer (2% donkey serum (116-4101, Thermo Fisher Scientific), 0.1% bovine serum albumin (BSA; A9418, Sigma-Aldrich), 0.01% Tween 20 (BP337-100, Thermo Fisher Scientific) in PBS) at room temperature.
9
Immunofluorescent Analysis of SiglecF+ and KLF4+ Cells
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Isolated cells from lavage were plated in a glass-bottom dish. After 2 h, the cells were washed with PBS followed by a 10-min Fc block. Then surface staining was done with anti-SiglecF-APC (BioLegend) tagged antibody. After 30 min, cells were fixed with 4% paraformaldehyde (#P6148; Sigma-Aldrich) and permeabilized with 0.05% Triton X-100 (#BP151–100; Thermo Fisher Scientific). After a wash with PBS/Tween-20 (#BP337–100; Thermo Fisher Scientific), cells were blocked with 5% BSA (Sigma-Aldrich) in PBS/Tw-20 for 1 h at room temperature. Afterward, cells were incubated with anti-KLF4 primary antibodies (#AF3158; R & D system) and incubated overnight at 4°C. The next day, slides were washed and incubated with the fluorescence-conjugated secondary antibody (AF546 donkey anti-goat 1:300, #A-11056; Invitrogen). Images were taken with a confocal microscope LSM880 (Zeiss) and analyzed by ImageJ.
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