Post-fixed spinal cords were cryo-protected in 15% and 30% sucrose (Sigma, S9378) in 1×PBS. Spinal cords were mounted in cryomolds (Tissue-Tek® Cryomold®, 420572) using compound (Tissue-Tek® O.C.T.™ ) and rapidly frozen to − 60 °C. Twenty-micrometer coronal sections were produced using a cryostat (Leica, CM1850, − 22 °C) and mounted on slides (VWR, SuperFrost® Plus, 48311-703). Sections were thawed, rehydrated in 1×PBS, blocked for 2 h at RT in blocking solution (0.3% Triton X-100 (Sigma, 93443), 5% normal goat serum (Serotec, 301104, 1×PBS and 0.01% sodium azide (Sigma, S-2002)). Primary antibody (Table 1 ) was added, and sections were incubated at 4 °C for 24 h. Sections were rinsed in 1×PBS followed by incubation in secondary antibody (Table 1 ) at RT for 1 h. Sections were incubated at RT for 20 min with nucleic acid stain (Hoechst 33258, Invitrogen™ H3569). Prior to confocal microscopy, the slides were rinsed and mounted using Mowiol (Sigma, 81381) and a cover slip (Marienfeld, 010243).
H3569
Manufactured by Thermo Fisher Scientific
Sourced in United States
The H3569 is a laboratory centrifuge product manufactured by Thermo Fisher Scientific. It is designed to separate different components within a liquid sample through the application of centrifugal force. The centrifuge provides consistent and reliable performance for a variety of laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescent mounting medium, by Agilent Technologies (2 mentions)
Ab10333, by Abcam (2 mentions)
Ab10353, by Abcam (2 mentions)
Ab6785, by Abcam (2 mentions)
Zen blue software, by Zeiss (2 mentions)
Normal goat serum (ngs), by Bio-Rad (1 mentions)
S9378, by Merck Group (1 mentions)
Superfrost plus, by Avantor (1 mentions)
Mowiol, by Merck Group (1 mentions)
Cm1850, by Avantor (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Cover slip, by Marienfeld (1 mentions)
Ab6668, by Abcam (1 mentions)
Sc 8429, by Santa Cruz Biotechnology (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Imagexpress micro confocal, by Molecular Devices (1 mentions)
Af806, by R&D Systems (1 mentions)
A27040, by Thermo Fisher Scientific (1 mentions)
P1951, by Merck Group (1 mentions)
A 11015, by Thermo Fisher Scientific (1 mentions)
13 protocols using h3569
1
Cryopreservation and Sectioning of Spinal Cords
2
Immunofluorescence Staining of HEK293 Cells
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This assay was performed as previously reported26 (link), with slight modifications. One day before the experiment, HEK293 cells were plated at a density of 5 × 104 cells/well in complete medium. The next day, cells were washed with PBS/0.1% BSA, fixed with paraformaldehyde 4% during 15 min and rinsed with PBS/0.1% BSA. Cell membranes were subsequently permeabilized with 0.1% Triton X100 during 5 min. After a washing step with PBS/0.1% BSA, cells were incubated with the primary monoclonal antibody Ab4E3 (ab10333, Abcam, 5 μg/ml, diluted with PBS/0.1% BSA) or with its isotopic control (Mouse Ig2a kappa Monoclonal, ab10353, Abcam, 25 μg/ml) for 90 min in the dark. Cells were washed twice and incubated for 60 min with goat anti-mouse IgG coupled to FITC (ab6785, Abcam, 1 μg/ml) and with DAPI (Hoechst 33258 pentahydrate (bis-benzimide), H3569, Invitrogen). Finally, cells were fixed a second time with paraformaldehyde 4% during 5 min. Fluorescence was analysed in fluorescent mounting medium (Dako) with a digital Evos microscope (AMG, Westburg).
3
Quantitative Analysis of Osteochondral Tissue
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The osteochondral tissue of interest was frozen at −20 °C, and the frozen samples were subjected to an overnight digestion process using papain solution (Solarbio, P8150). The content of DNA, glycosaminoglycans (GAGs), and hydroxyproline were quantified using the fluorometric Hoechst 33,258 assay (Invitrogen™, H3569), 1,9-dimethyl methylene blue (DMMB) assay, and l -hydroxyproline assay (acid hydrolysis method), respectively [[20] (link), [21] (link), [22] ].
4
Retinal Cell Immunostaining Protocol
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Isolated fixed retinas were permeabilized for 30 min in 0.5% Triton X-100 in PBS at RT. Retinas were then blocked in 10% goat serum, 1% bovine serum albumin, and 0.5% Triton X-100 in PBS for one hour at RT. Subsequently, retinas were incubated in goat anti-mouse F(ab) fragment (1:2000;ab6668, Abcam, Cambridge, UK) for one hour at RT. Mouse monoclonal anti-Brn3a (1:100; SC-8429, Santa Cruz Biotechnology, Dallas, TX) was diluted in blocking buffer and incubated on retinas overnight at 4 °C. Fluorescent-conjugated secondary antibody (1:500; Alexa Fluor 647, A21235, Invitrogen, Carlsbad, CA) was diluted in blocking buffer and incubated on retinas for two hours at RT. Bisbenzimide (1:100; Invitrogen H3569, Carlsbad, CA) was diluted in wash buffer and added to retinas for 20 min at RT. Four incisions were made in each retina cup to allow them to lie flat on a slide, and retinas were then mounted with Fluoromount-G (0100–01, SouthernBiotech, Birmingham, AL).
5
Immunofluorescence Staining of HUVECs
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For immunofluorescence staining, HUVECs were fixed using 4% paraformaldehyde (PFA) in HBSS+ for 10 min at room temperature. The fixative was aspirated, and the cells were rinsed once with HBSS+. Next, the cells were permeabilized for 2 min with 0.2% Triton X-100 in HBSS+ and washed once with HBSS+. The cells were blocked in 5% BSA in HBSS+ for 30 min and incubated with the primary antibody solution overnight at 4°C. Mouse anti-human CD144 (1:150; 555661, BD Biosciences, USA), sheep anti-human CD31 (1:150; AF806, R and D Systems, The Netherlands) and rabbit anti-human vWF (1:1000; A0082, Agilent Dako, USA) were used as the primary antibodies. The cells were washed with HBSS+, followed by an one-hour incubation with Hoechst (1:2000; H3569, Invitrogen, USA), rhodamine phalloidin (1:200; P1951, Sigma-Aldrich, The Netherlands) and the secondary antibody solution, containing Alexa Fluor 488-conjugated goat anti-mouse (1:250; R37120, ThermoFisher, USA), Alexa Fluor 488-conjugated donkey anti-sheep (1:250; A11015, ThermoFisher, USA) and Alexa Fluor 647-conjugated goat anti-rabbit (1:250; A27040, ThermoFisher, USA) antibodies. The cells were washed three times with HBSS+. High-quality Z-stack images of the stained cells were acquired using a high-content confocal microscope (Molecular Devices, ImageXpress Micro Confocal).
6
Immunofluorescence Staining of HEK293 Cells
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One day before the experiment, HEK293 cells were plated at a density of 105 cells/well in complete medium. The next day, cells were rinsed with PBS/0.1% BSA, fixed with 4% of paraformaldehyde during 15 min and rinsed with PBS/0.1% BSA. Cell membranes were subsequently permeabilized with 0.1% Triton X100 during 5 min. After a washing step with PBS/0.1% BSA, cells were incubated with the primary monoclonal antibody Ab4E3 (ab10333, Abcam, 5 μg/ml, diluted with PBS/0.1% BSA) or with its isotopic control (Mouse Ig2a kappa Monoclonal, ab10353, Abcam, 25 μg/ml) for 90 min in the dark. After two washing steps with PBS and PBS/0.1% BSA, respectively, cells were incubated for 60 min with goat anti-mouse IgG coupled to FITC (ab6785, Abcam, 1 μg/ml) and with DAPI (Hoechts 33258 pentahydrate (bis-bezimide), H3569, Invitrogen). Finally, the cells were fixed once again with 4% of paraformaldehyde during 5 min. Fluorescence was analyzed in fluorescent mounting medium (Dako) with a digital microscope Evos fluorescence (AMG).
7
FISH Protocol for Nuclear Foci Detection
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FISH experiments were performed as previously described [26 (link)]. Briefly, cells were fixed with PBS buffer containing 4% PFA (Electron Microscopy Science, 20 μl/well) for 20 min at room temperature and washed with 200 μl of PBS. Cells were then incubated overnight at 4°C with 70% Ethanol (60 μl/well). After PBS wash, cells were rehydrated with a solution of PBS containing 5mM MgCl2 for 15–30 min and then sequentially incubated with prehybridisation buffer (50 mM Phosphate buffer, 40% formamide, 2X SSC, 50 μl/well) for 15–30 min and hybridisation buffer (prehybridisation buffer with 0.2% BSA and 7% Dextran, 50 μl/well) containing 300 ng/ml of the (CAG)10‐Cy5 probe (5′Alexa 647‐TTCTTATTCTTCAgCAgCAgCAgCAgCAgCAgCAgCAgCAg3′) (Operon) overnight at 37°C. The washing steps consisted of prewarmed washing buffer (prehybridisation buffer + 0.2% BSA, 60 μl) for 15 min at RT followed by the addition of washing buffer (80 μl) and incubation for 30 min at 37°C. Cells were washed twice in PBS before nuclei counterstaining Hoechst 33258 (ThermoFisher®, H3569). Plates were stored at 4°C until analysis. Nuclear foci detection was performed using the Cellomics Array Scan VTI high‐content imaging system (Thermofisher®) with the 20X objective. Images were acquired in high‐resolution camera mode on two channels, and quantification was performed as previously described [26 (link)].
8
Immunohistochemical Labeling of GABAergic AII Amacrine Cells
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Briefly, 10-μm-thick cryosections were fixed in 4% paraformaldehyde for 20 min at room temperature and then incubated in 0.1% Triton X-100/PBS for 30 min at 37 °C. After incubation in 3% bovine serum albumin/PBS for 1 h at room temperature, the cryosections were incubated with a rabbit polyclonal antibody against GABAAR (Sigma-Aldrich catalog #A2052, RRID: AB_477652, 1:100) for 1 day at 4 °C to label GABAergic AII amacrine cells. An Alexa Fluor 555-conjugated donkey anti-rabbit IgG antibody (Thermo Fisher Scientific catalog #A-31572, RRID: AB_162543, 1:1,000) was used as the secondary antibody. The sections were counterstained with the nucleic acid stain Hoechst 33258 (Thermo Fisher Scientific catalog #H3569, RRID: AB_2651133, 1:2,000) in PBS. A laser scanning confocal microscope with a 63× oil-immersion objective lens (TCS SP8; Leica Microsystems) was used for imaging.
9
Immunostaining of hiPSC-derived Motoneurons
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Motoneurons derived from hiPSC were fixed with a solution containing 4% paraformaldehyde (PFA‐Euromedex®) for 8 min at room temperature. Cocultures were fixed vol:vol with a solution containing 8% paraformaldehyde (PFA‐Euromedex®, 15710) for 10 min at room temperature. Primary antibodies were diluted in PBS/1% BSA/0.2% Triton X‐100 and incubated overnight at 4°C. Appropriate secondary antibodies were added for 1 h at room temperature in the presence of Hoechst 33258 (ThermoFisher®, H3569). Primary and secondary antibodies are listed in the supporting information Material and Method Table S2 .
10
Stem Cell-Derived Beta Cell Proliferation Assay
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