After allowing the neurites to freely extend for 48 hours, the DRG neurons were pretreated with capsazepine (a TRPV1 antagonist, 10 μM) (Sigma-Aldrich) for 30 minutes. Thereafter, the neurons were treated and cultured for 24 hours as follows: (1) formaldehyde group (n = 5), DRG neurons were exposed to formaldehyde (Sigma-Aldrich) 10 μM; (2) menthol group (n = 5), DRG neurons were exposed to menthol (Sigma-Aldrich) 300 μM; (3) PD98059 + formaldehyde group (n = 5), PD98059 (an ERK1/2 inhibitor, 10 μM) (Cell Signaling Technology, Danvers, MA, USA) was added 30 minutes before formaldehyde (10 μM) exposure; (4) PD98059 + menthol group (n = 5), PD98059 (10 μM) was added 30 minutes prior to menthol (300 μM) exposure; (5) control group (n = 5), DRG neurons treated only with the TRPV1 receptor antagonist capsazepine (10 μM). Because TRPA1 and TRPV1 channels interact in neurons (Ruparel et al., 2011), capsazepine, a TRPV1 antagonist, was used to block the effects of TRPV1.
Formaldehyde
Manufactured by Cell Signaling Technology
Sourced in United States
Formaldehyde is a fixative chemical used in various laboratory applications. It functions by cross-linking and preserving the structural and chemical properties of biological samples, such as cells and tissues, for subsequent analysis and experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Tween 20, by Merck Group (2 mentions)
Formaldehyde, by Merck Group (1 mentions)
Menthol, by Cell Signaling Technology (1 mentions)
Menthol, by Merck Group (1 mentions)
Capsazepine, by Merck Group (1 mentions)
Capsazepine, by Cell Signaling Technology (1 mentions)
Pd98059, by Cell Signaling Technology (1 mentions)
Ab203591, by Abcam (1 mentions)
Dynabeads protein a, by Thermo Fisher Scientific (1 mentions)
Bioruptor pico, by Diagenode (1 mentions)
Non phospho active β catenin antibody, by Cell Signaling Technology (1 mentions)
Hiseq x ten pe150, by Illumina (1 mentions)
Anti phospho stat3 tyr705, by Cell Signaling Technology (1 mentions)
Mowiol, by Merck Group (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Lsm 880 airyscan confocal microscope, by Zeiss (1 mentions)
Nunc lab tek 2 chamber slide, by Thermo Fisher Scientific (1 mentions)
Eclipse ti2 inverted microscope, by Nikon (1 mentions)
Triton x, by Merck Group (1 mentions)
Dapi fluroshield, by Abcam (1 mentions)
9 protocols using formaldehyde
1
Investigating Menthol and Formaldehyde Effects on Neurons
2
Chromatin Immunoprecipitation (ChIP) Assay
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ChIP assays were carried out in terms of the manual of manufacturer (Cell Signaling Technology). Briefly, cells were cross-linked with 1% formaldehyde (Cell Signaling Technology), and formaldehyde was inactivated by the addition of 1M glycine. Chromatin extracts containing DNA fragments with an average size of 150–900 bp were immunoprecipitated using anti-KLF2 antibody (ab203591, 1:30, Abcam) or IgG (Cell Signaling Technology). The precipitated DNA was analyzed via real-time PCR.
3
Chromatin Immunoprecipitation of Active β-Catenin
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ChIP was performed according to the reported previously [56 (link)]. Briefly, about 2 × 106 TS cells were cross-linked with 16% formaldehyde (Cell Signaling Technology, 12,606) at final concentration of 1% at room temperature for 10 min and quenched with 1/10 volume of 1.25 M glycine for 15 min on ice. Cell lysate in lysis buffer III were sonicated using Bioruptor pico (Diagenode) and then incubated with 4 μg non-phospho (active) β-catenin antibody (Cell Signaling Technology, 8814) overnight at 4°Cwith rotation. Immunoprecipitated complexes were collected with 15 μl Protein A Dynabeads (Invitrogen, 10006D) for 1 h at 4 °C with rotation. Subsequently, beads were washed sequentially once with low-salt buffer, twice with high-salt buffer, once with LiCl buffer, twice with TE, and then eluted in 400 μl of elution buffer for 30 min at 65 °C with shaking. The eluates were incubation at 65 °C for 8 h to reverse the cross-linking. Next, eluates were treated with proteinase K for 1 h at 55 °C and then RNase A for 30 min at 37 °C before DNA was extracted and purified. The ChIP libraries were prepared according to the instruction manual using KAPA DNA HyperPrep Kits (Roche, KK8502) and then run on the Illumina sequencer Hiseq-Xten PE150. Primers for qPCR were listed in Additional file 1 : Table S1.
4
Leptin-Induced STAT3 Phosphorylation in AML12 Cells
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AML12 cells were treated with or without murine leptin (50 ng/mL) for 12 h and fixed in 1% formaldehyde (Cell Signaling Technology, Boston, USA). The chromatin immunoprecipitation (ChIP) assay was carried out according to the manufacturer’s protocol (Cell Signaling Technology, #9002). Rabbit immunoglobulin G (IgG) was used as a control for nonspecific immunoprecipitation of DNA and anti-Histone H3 (D2B12) XP® Rabbit mAb was used as a positive control. Anti-phospho-STAT3 (Tyr705) (Cell Signaling Technology, #9145) was purchased from Cell Signaling Technology. Immunoprecipitated protein–DNA crosslinking was reversed, and the DNA was purified for further qPCR analysis. ChIP enrichment efficiency was calculated for each sample (pStat3, IgG and H3 positive control) using the formula: percent input = 2% × 2 (CT 2% Input Sample − CT IP Sample). Alternatively, the enrichment efficiency of the pStat3 antibody was normalized to IgG (= 1). The qPCR products were separated with 1.5% agarose gel electrophoresis and imaged under ultraviolet light.
5
Synthesis of Gadolinium-Doped Boron Carbide Nanoparticles
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In this work, we used B4C nanopowder 99% pure from SkySpring Nanomaterials Inc., Houston, TX, USA; polyacrylic acid (PAA, 63% in dH2O, MW: 2000 Da) from Polyscience Inc.,Warrington, PA, USA; formaldehyde 16 % from Cell Signaling Technology (Danvers, MA, USA); Gd(NO3)3• H2O 99.99% pure from; NH4OH (NH3 28–30%) ; DiI (1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate); 3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyltetrazolium bromide; dimethyl sulfoxide; Hoechst 33342; and mowiol (all from Sigma Aldrich, DE St. Louis, MO, USA). dH2O was sterilized by filtration with a 0.2 μm pore size filter (Minisart, Sartorious AG, Gottingen, DE).
6
Fluorescent Labeling of Cultured Cells
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293- derived HFL, NLS2HF, and MYRHF cells were seeded at 5×105 cells per well in Nunc™ Lab-Tek™ II CC2™ Chamber (#154941, Thermo Scientific) in 500 μL complete DMEM medium. After 48 hours, cells were treated with 0.1 μM TAMRA (H-789) for 1 hr at 37 °C. Following treatment, cells were washed on the chamber twice with 1xPBS. 4% formaldehyde (#12606P, Cell Signaling) was added to fix cells for 15 min at room temperature, followed with 0.1% TritonX-100 permeabilization for 10min. After washing with 1xPBS for three times, 300nM Alexa Fluor® 647 Phalloidin and DAPI in 1xPBS were added to the cells for 30 min at room temperature. Cells were washed three times with 1xPBS, followed by adding 5uL of ProLong™ Gold. Slides were removed from the chamber and the cells covered overnight with coverslips (#7816, LabScientific). Cells were imaged on a Zeiss (Oberkochen, Germany) LSM 880 Airyscan confocal microscope.
7
Immunofluorescence Staining of Phleomycin-Treated Cells
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Cells were seeded onto Nunc® Lab-Tek® II Chamber Slides™ (Thermo Fisher Scientific, USA) and grown to 70% confluency. Cells were either left untreated or treated with phleomycin (1 µg/ml) for 1 hour. Medium was removed and cells were washed in PBS. Cells were then fixed in 4% formaldehyde (Cell Signaling Technology, USA) for 10 minutes. Fixed cells were washed twice for 5 minutes in PBS. Permeabilisation buffer containing 0.1% Triton-X (Sigma-Aldrich, USA) in PBS was added to each well and incubated for 10 minutes on a rotator at low speed. Permeablisation buffer was removed and cells were washed twice in PBS. Blocking buffer containing 0.1% Tween-20 (Sigma-Aldrich, USA) and 5% FBS was added and incubated at room temperature for 1 hour. Blocking buffer was removed and primary antibody was added and incubated overnight at 4°C. The next day, all wells were washed 3 times in blocking buffer and secondary antibody was added to each well. The chamber slide was wrapped in foil and incubated at room temperature for 1 hour. Wells were then washed 3 times in PBS and DAPI/Fluroshield (Abcam, UK) were added to the slide prior to coverslipping. Slides were imaged using a Nikon Eclipse Ti-2 Inverted Microscope at 40 × magnification with an exposure time of 100 ms.
8
Immunofluorescent Analysis of NUPR1 Expression
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Cells were seeded in a confocal dish (SPL Life Sciences, Gyeonggi-do, Korea) and then cultured in single (doxorubicin, or ATZ-502) and combination (doxorubicin and ATZ-502) treatments for 4 h. Immunofluorescent staining of cells was performed as previously described [35 (link),36 (link)]. Briefly, confocal dishes were fixed with the fix buffer consisting of 4% formaldehyde (Cell Signaling Technology, MA, USA) for 15 min at room temperature. After washing three times with wash buffer (Cell Signaling Technology, MA, USA), cells were then permeabilized with 100% cold methanol for 10 min at −20 °C and treated with blocking buffer (Cell Signaling Technology, MA, USA) for 1 h at ambient temperature. Primary antibody, anti-NUPR1 (NOVUS Biologicals, CO, USA, 1:200), for immunofluorescence was incubated overnight at 4 °C. After washing three times, cells were incubated with the secondary antibody Alexa Fluor 488 (Abcam, MA, USA, 1:1000) for 1 h under dark conditions. Each confocal dish was observed on a ZEISS LSM 800 confocal microscope (ZEISS, Jena, Germany). Single and 3D images were assessed in a ZEN system of ZEISS image software (ZEISS, Jena, Germany).
9
Neutrophil Cytoskeletal Visualization
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Neutrophils were allowed to crowd on the bacterial microarray for 60 min prior to fixation and permeabilization. The neutrophils were fixed with 4% formaldehyde (Cell Signaling Technology, Danvers, MA) in PBS for 15 min. The fixed cells were then permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) for 10 min. The permeabilized cells were then blocked with 10% normal goat serum for 1 h, followed by an incubation with a primary anti-β-tubulin antibody (2128; Cell Signaling Technology, Danvers, MA) at a dilution of 1:100 in 1% bovine serum albumin (BSA; Sigma-Aldrich) in 0.07% Tween 20 (Sigma-Aldrich). After an overnight incubation of the primary antibody at 4°C, secondary antibodies were incubated at a 1:1000 dilution in 1% BSA in 0.07% Tween 20. The actin filaments of the neutrophils were later stained with ActinRed 555 ReadyProbes reagent, according to the manufacturer’s protocol. Lastly, the nuclei were stained with a 300 nM 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific, Waltham, MA) solution in PBS for 5 min. The stained cells were mounted with ProLong Glass Antifade Mountant (Thermo Fisher Scientific) and imaged via epifluorescence (Nikon, Melville, NY) and confocal (Olympus, Tokyo, Japan) microscopy. Washing steps were performed three times between all incubations (except after blocking) with PBS for 5 min.
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