Methanol free formaldehyde

Manufactured by Thermo Fisher Scientific

Sourced in United States

Methanol-free formaldehyde is a laboratory reagent used in various applications, such as tissue fixation, histological staining, and protein cross-linking. It provides a reliable source of formaldehyde without the presence of methanol, which can be beneficial in certain experimental settings.

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Lab products found in correlation

Triton x 100, by Merck Group (5 mentions) Formaldehyde, by Thermo Fisher Scientific (4 mentions) M220 focused ultrasonicator, by Covaris (3 mentions) Acrylamide, by Merck Group (3 mentions) Sodium acrylate, by Merck Group (3 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Chip it express kit, by Active Motif (2 mentions) Ipure kit, by Diagenode (2 mentions) Lowcell chip kit protein a, by Diagenode (2 mentions) Corresponding rabbit igg, by Diagenode (2 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (2 mentions) Cell id cisplatin, by Standard BioTools (2 mentions) Fc receptor binding inhibitor, by Thermo Fisher Scientific (2 mentions) Cytof software, by Standard BioTools (2 mentions) Heavy metal conjugated antibodies, by Standard BioTools (2 mentions) Metal conjugated antibodies, by Standard BioTools (2 mentions) Cell id intercalator ir solution, by Standard BioTools (2 mentions) Nextseq 550, by Illumina (2 mentions) Protein a magnetic beads, by Thermo Fisher Scientific (2 mentions) Tapestation 4200, by Agilent Technologies (2 mentions)

Close spelling variants of this product

Methanol (2986 citations) Formaldehyde (433 citations) Methanol-free formaldehyde solution (4 citations)

60 protocols using methanol free formaldehyde

Nuclear Extraction and Fixation Protocol

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For human cell lines, nuclei were extracted as previously described (26 (link)) and resuspended in 150 μL of PBS. Then, 16% methanol-free formaldehyde (Thermo Fisher Scientific, PI28906) was added for fixation (final concentration: 0.1%) at room temperature for 3 min. The cross-linking reaction was stopped by addition of 12 μL 1.25 M glycine solution. Subsequently, nuclei were washed once with 150 μL antibody buffer (20 mM HEPES pH 7.6, 150 mM NaCl, 2 mM EDTA, 0.5 mM spermidine, 1% BSA, 1× protease inhibitors).
For NTT-seq on PBMCs and BMMCs, 16% methanol-free formaldehyde (Thermo Fisher Scientific, PI28906) was added for fixation (final concentration: 0.1%) at room temperature for 5 min. The cross-linking reaction was stopped by addition of 12 μL 1.25 M glycine solution. Subsequently, cells were washed twice with PBS. The permeabilization was performed by adding isotonic lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 3 mM MgCl₂, 0.1% NP40, 0.1% Tween-20, 1% BSA, 1× protease inhibitors) on ice for 7 min. Subsequently, 1 mL of cold wash buffer (20 mM HEPES pH 7.6, 150 mM NaCl, 0.5 mM spermidine, 1× protease inhibitors) was added, and cells were centrifuged at 800g for 5 min at 4°C.

Stuart T., Hao S., Zhang B., Mekerishvili L., Landau D.A., Maniatis S., Satija R, & Raimondi I. (2022). Nanobody-tethered transposition enables multifactorial chromatin profiling at single-cell resolution. Nature biotechnology, 41(6), 806-812.

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Evaluating DNA Damage Response to Small Molecule Treatments

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IEC-6 cells were seeded in 8-well chamber slides (Thermo Fisher Scientific) at 5 × 10⁴ cells per well and serum starved in 2% FBS medium overnight. Cells were treated with DMSO, 3 µM MMC, or the indicated concentration of 742 or 746 for 24 hours before washing once with cold PBS and fixation in 4% methanol free formaldehyde (Thermo Fisher Scientific) for 30 min on ice, washing with PBS and quenching of free aldehydes with a 50 mM NH₄Cl solution for 10 minutes. Cells were permeabilized using 0.25% Triton-X 100 (Sigma Aldrich) for 5 min on ice, washed with PBS, and blocked with blocking solution (PBS containing 1% BSA and 5% normal goat serum) for 1 hour on ice and incubated with the Phospho-Histone H2AX (Ser139) rabbit monoclonal antibody (Cell Signaling, 9718) diluted 1:800 in blocking solution overnight at 4°C. Cells were washed with PBS and incubated in the secondary Alexa Fluor 488 goat anti-rabbit IgG (H+L) antibody (Life Technologies, A-11034) for 45 minutes at room temperature. Cells were washed with PBS and mounted with VECTASHIELD mounting medium with DAPI (Vector Labs). Cells were examined for immunofluorescence using a Leica DM6000B upright microscope and counted as positive if more than five foci/nucleus were detected.

Dougherty M.W., Valdés-Mas R., Wernke K.M., Gharaibeh R.Z., Yang Y., Brant J.O., Riva A., Muehlbauer M., Elinav E., Puschhof J., Herzon S.B, & Jobin C. (2023). The microbial genotoxin colibactin exacerbates mismatch repair mutations in colorectal tumors. Neoplasia (New York, N.Y.), 43, 100918.

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Immunofluorescence Labeling of Cultured Cells

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Cells were grown to 80% confluence on cover slips in 12-well plates prior to fixation with 0.5 ml 4% methanol-free formaldehyde (Thermo Fisher Scientific) for 4 min at room temperature. Permeabilisation was achieved using 0.5 ml 100% ice-cold ethanol and incubation at −20°C overnight. Ethanol was removed and 1 ml wash buffer (1x TBS, 0.2% Triton X100 and 0.04% SDS, Sigma-Aldrich was added to the wells for 5 min. Cells were blocked in 1 ml blocking buffer (3% bovine serum albumin diluted in 1xTBS) for 1 h at room temperature. Cover slips were incubated with 60 μl primary antibody diluted in blocking buffer for 1 h at room temperature. Three washes were performed with 1 ml wash buffer for 5 min each. Secondary antibodies (Alexa Fluor 488 donkey anti-rabbit or Alexa Fluor 568 donkey anti-mouse, Thermo Fisher Scientific) were diluted in blocking buffer and 60 μl was added to each cover slip for 1 h at room temperature in the dark. Cells were washed as before prior to mounting the coverslips with mounting medium containing DAPI (Vectashield) on microscope slides (ThermoScientific) sealed with nail varnish at the edges. Slides were stored at 4°C in the dark until analysis. Images were analysed on the Zeiss Observer Z1 Microscope using a 40X oil objective or a 20X objective. Images were analysed using ImageJ software.

Taylor C., Mannion D., Mirand a F., Karaminejadranjbar M., Herrero-Gonzalez S., Hellner K., Zheng Y., Bartholomeusz G., Bast RC J.r, & Ahmed A.A. (2017). Loss of PFKFB4 induces cell death in mitotically arrested ovarian cancer cells. Oncotarget, 8(11), 17960-17980.

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Quantifying HIF-1α Stabilization in AT-MSCs

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AT-MSCs were plated in black 96-well clear-bottom plates (# 6005182, Perkin-Elmer) at a cell-density of 1 × 10⁴ cells/well and allowed to adhere overnight. Cells were cultured for 5 and 72 h in MM with 312.5, 625, and 1563 µM of DMOG, or 16, 32, 162, and 624 µM of baicalein, or with equal volumes of DMSO. The HIF-1α stabilization was quantified using the In-Cell ELISA Near Infrared Detection Kit (# 62201, Thermo Fisher Scientific) following the manufacturer’s instructions. Cells were fixed in 4% methanol-free formaldehyde (# 28906, Thermo Scientific), followed by washing, permeabilization, and blocking steps. HIF-1α was probed using mouse anti-human HIF-1α (1:50, # 610959, BD Biosciences), and an antibody for housekeeping protein rabbit polyclonal beta actin (2 µg/mL, # PA5-16914, Thermo Fisher Scientific). After overnight incubation at + 4 °C, AT-MSCs were washed with 1 × wash buffer before incubation with species-specific near-infrared DyLight-conjugated secondary antibody mix for 1 h at RT. After washing steps, Odyssey FC Imager (LI-COR) served to scan the plates with excitation/emission maxima of 692/712 nm for DyLight 680 Dye and 777/794 nm for DyLight 800 Dye. Measured signals were analyzed with Image Studio Software (LI-COR).

Abu-Shahba A.G., Gebraad A., Kaur S., Paananen R.O., Peltoniemi H., Seppänen-Kaijansinkko R, & Mannerström B. (2020). Proangiogenic Hypoxia-Mimicking Agents Attenuate Osteogenic Potential of Adipose Stem/Stromal Cells. Tissue Engineering and Regenerative Medicine, 17(4), 477-493.

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Visualizing Cell Adhesion Structures

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Cells were plated on collagen-I coated 4-chamber cell culture slide (BD Biosciences, Bedford, MA) 48 hours before fixation. Then, cells were fixed with 4% methanol-free formaldehyde (Thermo Scientific) for 15 min, permeabilized with 0.1% Triton X-100 for 10 min and blocked with image-iT FX Signal Enhancer (Life Technologies) for 30 min. Vinculin antibody (Sigma) was used to stain focal adhesion formations and actin filaments were stained by Alexa-Fluor 546-conjugated phalloidin (Invitrogen). ProLong® Gold Antifade Reagent with DAPI (Life Technologies) was used as a mountant. Images were taken by EVOS FL Auto microscope (Life Technologies).

Shiraishi T., Verdone J.E., Huang J., Kahlert U.D., Hernandez J.R., Torga G., Zarif J.C., Epstein T., Gatenby R., McCartney A., Elisseeff J.H., Mooney S.M., An S.S, & Pienta K.J. (2014). Glycolysis is the primary bioenergetic pathway for cell motility and cytoskeletal remodeling in human prostate and breast cancer cells. Oncotarget, 6(1), 130-143.

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Histological and Immunohistochemical Analysis of Tissue Samples

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Wet cell-cultured samples were fixed in 4% methanol-free formaldehyde (Thermo Fisher Scientific, USA) overnight at 4°C. Applying paraffinization process, samples were paraffinized. 5 micron thick sections were cut from the paraffinized samples for staining.
The sections were stained separately with hematoxylin and eosin (Thermo Fisher Scientific, USA), picrosirius red (ScyTek Lab, USA) and Safranin O (ScienCell Research Lab, USA) for their histological analysis following manufacturer protocols and/or our previously defined protocols [18 (link), 29 ].
The sections were stained separately with elastin (ab21610, abeam, USA), tropoelastin (ab21600, abeam, USA), vimentin (ab92547, abeam, USA) and α=smooth muscle actin (SMA) markers for their immunohistochemical analysis following manufacturer protocols and/or our previously defined protocols [18 (link), 29 ].
Following the procedures stated above, explanted samples were fixed, paraffinized, sectioned and stained separately with DAPI (Thermo Fisher Scientific, USA) for their immunofluorescent analysis [18 (link), 25 (link), 30 ].

Jana S., Bhagia A, & Lerman A. (2019). Optimization of polycaprolactone fibrous scaffold for heart valve tissue engineering. Biomedical materials (Bristol, England), 14(6), 065014.

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Super-resolution Imaging of tdPCP-EGFP

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For super-resolution imaging of tdPCP-EGFP at the transcription site, cells were nucleofected and seeded in 8-chamber coverglass samples as outlined above. Media were removed, cells were briefly rinsed with RT 1×PBS and then fixed with 4% methanol-free formaldehyde (Thermo Fisher) in 1×PBS for 10min at RT, washed with 1×PBS and quenched with 50mM NH₄Cl in 1×PBS for 5 minutes. Following 3×10 minute washes in 1×PBS, cells were permeabilized for 10 minutes with 0.5% Triton X-100 in 1×PBS, and blocked for 10 minutes with Image-iT FX Signal Enhancer reagent (Cell Signaling Technology) and 1.5hrs with 5% w/v BSA in 1×PBS. Then the cells were stained with ~20nM GFP nanobody (Chromotek, GFP-Trap) that was labeled using Alexa 647-NHS (Life Technologies) at ~1:1 dye:nanobody ratio in 5% BSA/×PBS. After 3×5 minute washes in 1×PBS, cells were imaged in photo-switching buffer that contained 75mM HEPES-KOH pH 7.5, 0.9% w/v Glucose, 55mM Potassium Glutamate, 100mM β-Mercaptoethylamine and a mixture of glucose oxidase and catalase enzymes.

Li J., Dong A., Saydaminova K., Chang H., Wang G., Ochiai H., Yamamoto T, & Pertsinidis A. (2019). Single-molecule nanoscopy elucidates RNA Polymerase II transcription at single genes in live cells. Cell, 178(2), 491-506.e28.

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Live-cell Imaging with SiR Staining

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Cells were nucleofected, seeded in 8-chamber coverglass samples and stained with SiR as outlined above. Media were then removed, cells were briefly rinsed with 1×PBS pre-warmed at 37°C and then fixed with pre-warmed 4% methanol-free formaldehyde (Thermo Fisher) in 1×PBS for 10min at 37°C and washed with 1×PBS at RT. Imaging was performed in buffer that contained 75mM HEPES-KOH pH 7.5, 0.9% w/v Glucose, 55mM Potassium Glutamate, 1mM methyl viologen, 1mM ascorbic acid and a mixture of glucose oxidase and catalase enzymes.

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Immunofluorescence Staining of Cells

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Thin glass cover slips were ethanol and flame sterilised. The cells were seeded on the glass cover slips for 24 h. The unattached cells were washed away with PBS and cells were fixed with 4% methanol-free formaldehyde (Thermo scientific) for 20 minutes at room temperature. Cells were permeabilised with 0.01% Triton-X for 5 minutes at room temperature, washed with PBS. Nonspecific sites were blocked using 10% BSA for 1 h. Then cells were stained using rhodamine conjugated Phalloidin (Sigma), followed by nucleus staining using 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen). Images were captured using confocal microscope (Nikon Eclipse TE2000-U).

Beer M., Kuppalu N., Stefanini M., Becker H., Schulz I., Manoli S., Schuette J., Schmees C., Casazza A., Stelzle M, & Arcangeli A. (2017). A novel microfluidic 3D platform for culturing pancreatic ductal adenocarcinoma cells: comparison with in vitro cultures and in vivo xenografts. Scientific Reports, 7, 1325.

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Transferrin Uptake Visualization

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Cells grown to 100% confluence were washed once with PBS including Ca 2+ and Mg 2+ (PBS + ) and incubated for 10 min with 5µg/ml Transferrin, Alexa Flour 488 conjugate (Life Technologies, Carlsbad, CA) at 37°C under 5% CO2. Cells were washed once with room temperature acid wash medium and then fixed with 4% methanol free formaldehyde (Thermo Fisher Scientific, Waltham, MA) in PBS for 10 min. Samples were washed twice with PBS and sealed with FluorSave mounting medium. Transferrin uptake was visualized by confocal microscopy.

Kretschmer M., Mamistvalov R., Sprinzak D., Vollmar A.M., & Zahler S. (2022). Matrix stiffness regulates Notch signaling activity in endothelial cells.

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