ICD dosing was determined by in vitro CRT exposure and HMGB1 release.63 (link) Briefly, BPD6 cells treated with MIT alone or MIT+CEL (5:1 molar ratio), then harvested, PBS washed and fixed in 0.25 % PFA in PBS for 5 min. Cells were washed twice in PBS, and a primary antibody, diluted in cold blocking buffer, was added for 30 min. After three washes in cold PBS, cells were incubated for 30 min with the appropriate FITC-conjugated secondary antibody diluted in a cold blocking buffer. Cells were fixed with 4 % PFA for 20 min and then nuclei were stained with Hoechst 33432 (ThermoFisher Scientific), followed by Confocal imaging. For intracellular HMGB1 staining, cells were washed with PBS, fixed with 0.4 % PFA for 20 min, permeabilized with 0.1 % Triton X-100 for 10 min, rinsed three times with PBS, and nonspecific binding sites were blocked with 10 % goat serum in PBS for 30 min. A primary antibody for HMGB1 was added for 1 h. Subsequently, cells were washed three times with PBS and incubated for 30 min with a secondary antibody for fluorescence imaging.
Hoechst 33432
Manufactured by Thermo Fisher Scientific
Sourced in United States
Hoechst 33432 is a fluorescent dye used for nucleic acid staining. It binds to the minor groove of DNA and emits a blue fluorescence when excited by ultraviolet light.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluorophore, by Thermo Fisher Scientific (3 mentions)
Operetta cls system, by PerkinElmer (3 mentions)
Anti f4 80, by Bio-Rad (2 mentions)
Ab1903, by Abcam (1 mentions)
Cy3 conjugated anti mouse igg secondary antibody, by Jackson ImmunoResearch (1 mentions)
Mab4400, by Merck Group (1 mentions)
Shandontm immu mounttm, by Thermo Fisher Scientific (1 mentions)
880 confocal microscope, by Zeiss (1 mentions)
Zen lite software, by Zeiss (1 mentions)
Anti o4, by R&D Systems (1 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Wheat germ agglutinin alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Rat tail collagen, by Merck Group (1 mentions)
Fv1200 confocal microscope, by Olympus (1 mentions)
Facsaria iiiu, by BD (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Perm wash buffer, by BD (1 mentions)
Alexa488 and alexa594 conjugated secondary antibodies, by Thermo Fisher Scientific (1 mentions)
11 protocols using hoechst 33432
1
Quantifying HMGB1 Release Dynamics
2
Immunofluorescent Staining of HEK-293T Neurons
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HEK-293T neurons growing on glass coverslips were fixed in 4% paraformaldehyde for 15 min and then washed twice with PBS containing 20 mM glycine before permeabilization with the same buffer containing 0.2% Triton X-100 (5 min incubation). Cells were treated for 1 h with PBS containing 1% bovine serum albumin. To detect the expression of NR1-Rluc, cells were labeled with a mouse anti-Rluc antibody (1/100; MAB4400, Millipore, Burlington, MA, USA) and subsequently treated with Cy3-conjugated anti-mouse IgG secondary antibody (1/200; 715-166-150; Jackson ImmunoResearch, West Grove, PA, USA) (1 h each). The expression of CB1R-YFP was detected by the YFP’s own fluorescence. The presence of α-syn fibrils was detected with a mouse monoclonal anti-human α-synuclein antibody (1/300; ab1903, Abcam). Phalloidin was detected with an AF488 pre-stained anti-phalloidin probe (1/200; A12379, ThermoFisher, Waltham, MA, USA). Nuclei were stained with Hoechst33432 (1/100 from stock 1 mg/mL; Thermo Fisher, Waltham, MA, USA). The samples were washed several times and mounted on glass slides with ShandonTM Immu-MountTM (9990402; ThermoFisher, Waltham, MA, USA). Samples were observed under a Zeiss 880 confocal microscope (Carl Zeiss, Oberkochen, Germany) equipped with an apochromatic 63× oil-immersion objective (N.A. 1.4) and with 405 nm, 488 nm, and 561 nm laser lines.
3
Immunocytochemical Analysis of Oli neu Cells
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Oli neu cells were plated on poly-l -Lysine-coated 12 mm-coverslips at a density of 3 × 104
cells/well. After 24 h or 120 h, cells were fixed with 4% paraformaldehyde for 20 min at RT and washed three times in PBS.
The cells were permeabilized by incubation in PBS containing 0.1% Triton X-100, 10% NGS (Thermo Fischer, Walthman, MA,
USA), 1% BSA (Sigma-Aldrich, St. Louis, MO, USA) for 45 min at RT. The cells were then incubated overnight at 4 °C
with anti-O4 (10 µg/mL, R&D Systems, Inc. NE Minneapolis, MN, USA) and anti-Claudin-11 (1:100, Immunological
Science, Milan, Italy) antibodies, diluted in PBS with 0.1% Triton X-100, 1% NGS and 1% BSA. The cells were then washed
in PBS and incubated for 1 h and 30 min at RT with goat anti-mouse Alexa 594-conjugated and goat anti-rabbit Alexa
488-conjugated (Immunological Science) secondary antibodies, diluted 1:100 in PBS with 0.1%, 1% NGS and 1% BSA. Finally,
cells were incubated with Hoechst 33432 (1:1000 in PBS, Thermo Scientific, Waltham, MA, USA) for 10 min at RT, for the
nuclei counterstaining. At the end, coverslips were fixed on microscope slides with a PBS-glycerol (3:1; v/v) solution.
The images were acquired with a Zeiss fluorescence microscope through the Zen lite software (Zeiss, Oberkochen, Germany).
cells/well. After 24 h or 120 h, cells were fixed with 4% paraformaldehyde for 20 min at RT and washed three times in PBS.
The cells were permeabilized by incubation in PBS containing 0.1% Triton X-100, 10% NGS (Thermo Fischer, Walthman, MA,
USA), 1% BSA (Sigma-Aldrich, St. Louis, MO, USA) for 45 min at RT. The cells were then incubated overnight at 4 °C
with anti-O4 (10 µg/mL, R&D Systems, Inc. NE Minneapolis, MN, USA) and anti-Claudin-11 (1:100, Immunological
Science, Milan, Italy) antibodies, diluted in PBS with 0.1% Triton X-100, 1% NGS and 1% BSA. The cells were then washed
in PBS and incubated for 1 h and 30 min at RT with goat anti-mouse Alexa 594-conjugated and goat anti-rabbit Alexa
488-conjugated (Immunological Science) secondary antibodies, diluted 1:100 in PBS with 0.1%, 1% NGS and 1% BSA. Finally,
cells were incubated with Hoechst 33432 (1:1000 in PBS, Thermo Scientific, Waltham, MA, USA) for 10 min at RT, for the
nuclei counterstaining. At the end, coverslips were fixed on microscope slides with a PBS-glycerol (3:1; v/v) solution.
The images were acquired with a Zeiss fluorescence microscope through the Zen lite software (Zeiss, Oberkochen, Germany).
4
Doxorubicin-Induced HMGB1 Release
5
Cellular Uptake of Fluorescent Nanoparticles
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LabTek #1.5 borosilicate 8-well chamber slides were coated with 300 μL of rat tail collagen (Millipore Sigma) at a concentration of 50 μg/mL in 0.02N acetic acid for 5 minutes at room temperature. Chamber slides were washed once with PBS and air-dried for 5 minutes. 8,000 GBM22 cells were seeded per well in 300 μL media and allowed to adhere overnight before treatment with 5 μg/mL fluorescent NPs for 24 hours. Cells were carefully washed with HBSS on ice and incubated with 4 μg/mL wheat germ agglutinin-Alexa fluor 555 (Thermo Fisher) in ice-cold HBSS for 2 minutes. Cells were washed once with ice-cold PBS without incubation to remove excess wheat germ agglutinin and subsequently washed with PBS twice for 5 minutes before fixation with 4% paraformaldehyde (Pierce) for 15 minutes at room temperature. The cells were washed three more times with PBS and incubated with 4 μM Hoechst 33432 (Thermo Fisher) for 5 minutes at room temperature. Cells were imaged using an Olympus FV1200 confocal microscope with a 100x oil immersion objective. Images were pseudocoloured using FIJI.
6
Cell Cycle Analysis of Murine Hematopoietic Stem Cells
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Ki67 (BD Biosciences, Cat# 558617) and Hoechst 33432 (Invitrogen, Cat# H3570) were used for cell cycle analysis of fixed cells from C57BL/6 mice. A total of 4×106 BMMNCs/sample were stained with anti-CD150-APC, anti-CD48-FITC, anti-lineage (CD4, CD8a, Gr-1, Mac-1, Ter-119, B220)-PerCP-Cy5.5, anti-c-Kit-APC-Cy7, and anti-Sca-1-PE-Cy7 antibodies. To stain samples after 5-FU treatment, Mac-1 was excluded from the antibody cocktail. Stained samples were centrifuged at 340 × g and 4 °C for 5 min. To analyze HSCs derived from mice after in vivo 2-NBDG administration, anti-CD150-PE and anti-CD48-Alexa Fluor700 antibodies were alternatively used to sort and purify HSCs with high or low NBDG uptake. These HSCs were then subjected to Ki67/Hoechst staining. Next, 250 µL of BD Cytofix/Cytoperm (BD Biosciences, Cat# 555028) was added, and samples were incubated for 20 min at 4 °C for fixation. Fixed cells were centrifuged and washed twice at 340×g at 4 °C, with 1 mL BD Perm/Wash buffer (BD Biosciences, Cat# 554723) diluted 10-fold. Each sample was stained with 10 µL of Ki67-Alexa Fluor555 or Ki67-eFlour660 (for 2-NBDG stained HSC) antibodies for 1 h at RT, shaded from light. Ki67-stained cells were centrifuged and washed twice at 340 × g and 4 °C with PBS. Samples were resuspended in 500 µL of PBS + 10 µg/mL Hoechst 33432, filtered, and analyzed with the BD FACSAria IIIu instrument.
7
Immunofluorescence Analysis of Tumor Sections
8
Histological Analysis of Liver Apoptosis and Inflammation
9
Immunofluorescence Staining of Cultured Cells
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Cultured cells were fixed in 4% paraformaldehyde for 15 min, at RT, and subsequently permeabilized and blocked in Odyssey LiCor Blocking Buffer containing 0.2% Triton X-100. Primary antibodies were incubated overnight at 4 °C (anti-ITM2A polyclonal AF4876 and 14407-1-AP, monoclonal non-commercial provider Yumab: Yu093-G04, Yu147-A01, Yu147-E02, Yu147-H07, anti-EmGFP A31852 or A11122, anti-HA 901509), and appropriate secondary antibodies conjugated with Alexa fluorophores (Invitrogen) and Hoechst 33432 (Invitrogen) for nuclei staining were subsequently used for 2 h at RT. Images were acquired on a Perkin Elmer Operetta CLS system.
10
Immunocytochemistry of NHP-Derived BECs
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Immunocytochemistry was conducted on NHP-derived BECs following mono-culture conditions. Cultured cells were fixed in 4% p-formaldehyde for 15 min, at room temperature, and subsequently permeabilized and blocked in Blocking Buffer Odyssey LiCor containing 0.2% Triton X-100. Primary antibodies were incubated overnight (Anti-Claudin 5 Monoclonal (4C3C2) 1:200 #35-2500; Anti-ZO-1 Polyclonal, 1:200 #61-7300; Anti-Occludin Monoclonal (OC-3F10), 1:100 #33-1500, ThermoFisher; Anti-VE Cadherin Polyclonal, 1:500 #ab33168, Abcam, Amsterdam, The Netherlands), and appropriate secondary antibodies conjugated with Alexa fluorophores (Invitrogen) and Hoechst 33432 (Invitrogen) for nuclei staining were subsequently used. Images were acquired on a Perkin Elmer Operetta CLS™ system.
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