Freshly dissected mouse bones were fixed with 4% paraformaldehyde (PFA) (15,710 S, Electron Microscopy Sciences) for 4 hr at 4 °C. After samples were washed with PBS, decalcification was processed with 0.5 M EDTA for 5 days. Samples were incubated with infiltration solution (20% sucrose (S7903, Sigma) plus 2% polyvinylpyrrolidone (Sigma, 9003-39-8) in PBS) until they sank to the bottom of the tube. Embedding was performed with OCT (Sakura Finetek,4583) and samples were stored at −80 °C. The samples were sectioned at 10 μm in thickness using a Leica cryostat. Frozen sections were thawed at room temperature and rehydrated with PBS, permeabilized with 0.5% Triton X-100 in PBS for 15 min at room temperature, and blocked for 1 hr with 5% BSA in PBS (blocking buffer). Primary antibodies were freshly diluted in blocking buffer and were incubated with the slices overnight at 4 °C. After washing three times with PBS, secondary antibodies (1:2000 dilution with blocking buffer) were incubated for 1 hr at room temperature. Samples were then washed and mounted with antifade mounting solution with DAPI (Life technologies, P36941). Imaging was employed using a Zeiss Axioscan 7 Slide Scanner.
P36941
Manufactured by Thermo Fisher Scientific
Sourced in Japan
The P36941 is a laboratory instrument designed for the measurement and analysis of samples. It is a core product within the Thermo Fisher Scientific portfolio, providing essential functionality for scientific research and testing applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700, by Zeiss (2 mentions)
Lsm800 confocal microscope, by Zeiss (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
S7903, by Merck Group (1 mentions)
Axioscan 7 slide scanner, by Zeiss (1 mentions)
Cryostat, by Leica (1 mentions)
Sucrose, by Merck Group (1 mentions)
A11034, by Thermo Fisher Scientific (1 mentions)
A12380, by Thermo Fisher Scientific (1 mentions)
A12379, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent with 4 6 diamidino 2 phenylindole, by Thermo Fisher Scientific (1 mentions)
A31571, by Thermo Fisher Scientific (1 mentions)
Target retrieval buffer, by Agilent Technologies (1 mentions)
A11006, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade mounting media with dapi, by Thermo Fisher Scientific (1 mentions)
Rat anti ollas l2, by Novus Biologicals (1 mentions)
A11008, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
D3570, by Merck Group (1 mentions)
Sab4200007, by Merck Group (1 mentions)
8 protocols using p36941
1
Cryosectioning and Immunostaining of Mouse Bones
2
Immunofluorescence Staining of Paraffin-Embedded Tissue
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Paraformaldehyde-fixed tissue samples were embedded in paraffin and 7-μm sections were stained with hematoxylin and eosin or 0.1% picrosirius red. 25 (link) For immunofluorescence staining, sections were treated for antigen retrieval, blocked with 5% normal goat serum, and then incubated with primary antibodies at 4°C overnight, followed by incubation with fluorochrome-conjugated secondary antibodies (A-11007; Life Technologies/Thermo Fisher Scientific, Bartlesville, OK) at 37°C for 1 hour. Finally, the slides were mounted with antifade reagent containing DAPI (4',6-diamidino-2-phenylindole; P36941; Life Technologies). The following primary antibodies were used: Mac2 (14-5301-81; eBioscience, San Diego, CA), CD4 (550280; BD Biosciences, San Jose, CA), and CD8 (558733; BD Biosciences).
3
Quantifying DNA Damage Response in U2OS Cells
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U2OS cells were transfected with 100 nM scrambled control or XRCC1 siRNA; after 48 h, the cells were plated in eight-chamber slides, incubated overnight and then treated with HU and/or ATRi as indicated. The cells were then fixed with 4% paraformaldehyde for 15 min, followed by permeabilization with 0.5% Triton X solution in phosphate-buffered saline (PBS) for 30 min. Subsequently, after blocking with 3% bovine serum albumin (BSA) solution in PBS for 1 h, the cells were incubated with anti-phosphoserine H2A.X antibody (Cell Signaling Technology, #9718) or anti-XRCC1 antibody (#MS-434-P0, Thermo Scientific) diluted 1:500 in PBS for 2 h at room temperature. After washing with PBS, the cells were incubated with Alexa Fluor secondary antibody (1:500). After the final wash, the slides were dried for 5–10 min at 37°C and mounted with mounting media with DAPI (Invitrogen, P36941) and coverslips. Samples were observed under 60× oil immersion lens, and images were captured from at least 10 random fields for each sample. Cells were marked positive if they contained >10 foci.
4
Immunofluorescence Imaging of Mouse Cochlea
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Cochleae from adult mice were prepared and examined as described previously (85 (link), 86 (link)). All images were acquired with a confocal microscope (LSM 700 or 710, Zeiss). Samples were stained with phalloidin (A12379 or A12380, Invitrogen) or with antibodies for other markers. The following primary antibodies were used: anti–myosin VI (1:400; 25-6791; Proteus Bioscience), anti–myosin VIIa (5 μg/μl;138-1-s, Developmental Studies Hybridoma Bank), anti-Tuj1 (1:250; 801201, BioLegend), anti-pERK (1:400; 9101S, Cell Signaling Technology), or anti-Ctbp2 (1:500; 612044, BD Transduction). Anti-rabbit Alexa Fluor 488 (1:400; A11034) and anti-mouse Alexa Fluor 647 (1:400; A31571) secondary antibodies were purchased from Invitrogen. ProLong gold antifade reagent with 4′,6-diamidino-2-phenylindole (P36941, Invitrogen) was used to counterstain nuclei.
5
Subcellular Localization of IRF3 by HA-S273R
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3D4/21 cells were transfected with the HA-vector or HA-S273R for 24 h. Cells were fixed with 4% paraformaldehyde for 20 min at room temperature. After 3 washes with ice-cold PBS, cells were permeabilized with 0.1% Triton X-100 for 3 min; 5% bovine serum albumin (BSA) in PBST was used as the blocking buffer. After blocking at room temperature for 1 h, cells were incubated with mouse anti-IRF3 (10949S, Cell Signaling Technology, USA) and rabbit anti-HA tag (12698S, Cell Signaling Technology, USA) antibody at 4 °C overnight and then with Alexa Fluor 488-conjugated affinipure goat anti-mouse IgG (H + L) (1:100, P03S19S, Gene-Protien Link, China) and Alexa Fluor 594-conjugated affinipure goat anti-rabbit IgG (H + L) (1:100, P03S19L, Gene-Protien Link, China). The cells were observed by Nikon A1 laser confocal microscopy (Japan) after DAPI (P36941, Invitrogen) nuclear staining.
6
Immunofluorescent Analysis of RA Synovium
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Formalin fixed, paraffin embedded sections of human RA synovial membranes were single‐stained for either Gal‐3 or PD‐1. First, tissue sections were deparaffinated and rehydrated in Xylene and decreasing concentrations of ethanol, before demasking proteins in near‐boiling Target Retrieval buffer (DAKO, S2369, pH 6). Tissue sections were blocked in 10% donkey serum and single‐stained for PD‐1 (20 μg/mL murine anti‐human PD‐1 antibody (clone: NAT105, Abcam, ab52587), secondary antibody: AF488 Donkey anti‐mouse IgG (Jackson ImmunoResearch, 715‐546‐151) or Gal‐3 (10 μg/mL rat anti‐human Galectin‐3 antibody (clone: M3/38, MABT51)), secondary antibody AF488 AffiniPure Donkey anti‐rat IgG (Jackson ImmunoResearch, 712‐546‐153). For mounting and DAPI staining mounting media was applied (Invitrogen, P36941). Stained synovial membranes were examined using a Zeiss LSM800 confocal microscope. ImageJ was used to collect the signal from the z‐stacks obtained on the microscope into one image with maximum signal of the section.
7
Immunofluorescence Staining of Transfected Cells
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Cells were seeded and transfected on glass coverslips, 24 h post-transfection, cells were fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.1% Triton X-100 for 5 min. cells were blocked and incubated with primary antibodies against HA tag (Rabbit anti-HA C29F4, CST, Cat# 3724, dilution 1:500 v/v) and OLLAS tag (Rat anti-OLLAS L2, Novus Biologicals, Cat# NBP1-06713, dilution 1:100 v/v) for 2 h at room temperature. After washes, cells were stained with indicated fluorophore-conjugated secondary antibodies (Thermo Fisher, A-11008, A-11006) for another 1 h at room temperature. After wash, coverslips were mounted by ProLong Gold antifade mounting media with DAPI (Thermo Fisher, P36941). Images were taken by an LSM 880 confocal microscope (ZEISS).
8
Immunofluorescence Staining of DVL Proteins
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8 × 105 cells were seeded onto coverslips (12mm) in a 60mm tissue culture dish. The cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature, followed by a wash with PBS for 5 minutes, a quench step with 50mM ammonium chloride (NH4Cl) in PBS for 5 minutes with an additional 5 minutes PBS wash. The coverslips were blocked with 5% Bovine serum albumin (BSA) in PBS (blocking buffer) for 30 minutes, followed by an hour incubation with the following primary antibodies in 5% BSA in PBS: DVL-1 (D3570; Sigma) and DVL-3 (SAB4200007; Sigma). The samples were rinsed 3 times with PBS and then incubated with secondary antibodies purchased from ThermoFisher scientific (Alexa flour 568 #A11036, Alexa fluor 647 #A21235 and Alexa fluor phalloidin 488 #A12379 from Thermo Scientific) for 1 hour at room temperature. The samples were rinsed several times in PBS for 5 minutes each and then mounted with prolong gold antifade mounting solution with DAPI (P36941, Thermo Scientific), then cured overnight at room temperature and stored at −20°C until imaged. The samples were imaged using a laser scanning confocal microscope Nikon T-1E with a 60x objective and NIS software.
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