Paraformaldehyde (pfa)

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Paraformaldehyde is a solid, white polymeric form of formaldehyde. It is a commonly used fixative and preservative in laboratory applications.

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129 protocols using paraformaldehyde (pfa)

Raman Spectroscopy Analysis of Erlotinib Effect on Colon Cancer Cells

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The colon cancer cell lines SW-48, HT-29, and SW-480 were purchased from American Type Culture Collection. Cells were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies, Darmstadt, Germany) supplemented with 10 % fetal bovine serum (Life Technologies, Darmstadt, Germany), 2 mM l-glutamine, and 5 % penicillin–streptomycin, and were incubated at 37 °C in a 10 % CO₂ atmosphere. Cells were subcultured to 80 % confluence, detached by trypsin–EDTA (0.25 %) (Gibco trypsin solution, Life Technologies, Darmstadt, Germany), centrifuged at 1500 rpm for 3 min and diluted to 10 %, then seeded again in culture medium. Raman measurements were performed on cells grown on CaF₂ windows (Korth Kristalle, Kiel, Germany) to avoid Raman scattering from regular glass slides. Cells were incubated with erlotinib (Tarceva; Roche, Switzerland) at 10 μg/ml at 37 °C in 10 % CO₂ for 12 h. Subsequently, cells were fixed in 4 % paraformaldehyde (VWR International, Darmstadt, Germany) and then submerged in phosphate-buffered saline (Life Technologies, Darmstadt, Germany).

Yosef H.K., Mavarani L., Maghnouj A., Hahn S., El-Mashtoly S.F, & Gerwert K. (2015). In vitro prediction of the efficacy of molecularly targeted cancer therapy by Raman spectral imaging. Analytical and Bioanalytical Chemistry, 407(27), 8321-8331.

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Flow Cytometry-Based ORFV Infectivity Assay

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The infectivity of the ORFV samples was determined by a flow cytometry assay using a Guava easycyte HT (Merck Millipore, Darmstadt, Germany). The procedure was adapted from Lothert et al. [14 (link)], however, the washing procedure was omitted. Instead, after centrifugation, the cell pellets were resuspended with PBS, containing 1% paraformaldehyde, 2% FCS, and 2 mM EDTA (VWR International, Radnor, PA, USA). The readout was conducted within 48 h. The relative error between samples in one batch was below 10%. The effect of PEG on the ORFV infectivity was assumed to be neglectable, proven by full recoveries of infectious ORFV in previous experiments [13 (link),14 (link)]. The presence of all tested buffers and salts (Na₂SO₄, KCl, NaCl, NaNO₃, MgSO₄, or MgCl₂) applied in the SXC experiments had no effect on the virus infectivity (Supplementary Material S1) or the assay (Figure S1).

Eilts F., Lothert K., Orbay S., Pagallies F., Amann R, & Wolff M.W. (2022). A Summary of Practical Considerations for the Application of the Steric Exclusion Chromatography for the Purification of the Orf Viral Vector. Membranes, 12(11), 1070.

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Immunofluorescence Staining of Myc-Tagged Proteins

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For DAPI staining, cells were fixed with 70% cold ethanol, washed with PBS, and resuspended in PBS + 0.2 µg/ml DAPI (Sigma-Aldrich).
Immunofluorescence microscopy was performed as described previously (Hagan and Hyams, 1988 (link)). In brief, 10 ml of exponentially growing culture was fixed with freshly prepared 30% paraformaldehyde (294474L; VWR) in PEM buffer (0.1 M Pipes, 2 mM EGTA, and 1 mM MgSO₄, pH 6.9) plus glutaraldehyde (G6257; Sigma-Aldrich) at a final concentration of 0.2%, followed by 1-h incubation at room temperature. After washing three times with PEM, the cell wall was digested during 1 h at 37ºC with 2.5 mg/ml zymolyase 20T (120491-1; AMSBIO) in PEMS (PEM + 1.2 M sorbitol); cells were permeabilized with 1% Triton X-100 and quenched with 1 mg/ml sodium borohydride. After washing twice with PEM, cell pellet was resuspended in PEMBAL buffer (PEM buffer, 0.1% sodium azide, and 1% BSA) before antibody incubation. Primary antibody was mouse anti-Myc (9E10; Santa Cruz), used at a dilution of 1:100 for mts2-8Myc and 1:200 for mts4-13Myc. The secondary antibody was goat Alexa Fluor 488–tagged anti–mouse (A11029l; Invitrogen, Molecular Probes), used at a dilution of 1:1,000.

Salas-Pino S., Gallardo P., Barrales R.R., Braun S, & Daga R.R. (2017). The fission yeast nucleoporin Alm1 is required for proteasomal degradation of kinetochore components. The Journal of Cell Biology, 216(11), 3591-3608.

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Cochlear Tissue Preparation for Electron Microscopy

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After 7 days of culture, cochleae were fixed overnight in 4% paraformaldehyde (VWR) in PBS at 4 °C. NCs, and two control DCs that were not perfused with hWJCs, were stained for 24 hours with 2% osmium tetroxide (OT), because OT embeds between lipids, and produces a strong electron backscatter signal when bombarded by an electron beam. Afterward, OT-stained samples were washed three times for 10 minutes in PBS. All samples were gradually dehydrated with ethanol and cleared in xylene, before being embedded with paraffin. Samples were sectioned to a thickness of 10 μm using a microtome (Leica, Buffalo Grove, IL, USA) and mounted on SuperFrost glass slides (Thermo Fisher Scientific, Waltham, MA, USA).

Mellott A.J., Shinogle H.E., Nelson-Brantley J.G., Detamore M.S, & Staecker H. (2017). Exploiting decellularized cochleae as scaffolds for inner ear tissue engineering. Stem Cell Research & Therapy, 8, 41.

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Quantifying Intracellular Lipid Uptake

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Initially, apical-in organoids were incubated with 5 mM EDTA in PBS for 1 h on a shaking platform at 4°C, in order to remove the surrounding Matrigel. Both apical-in and apical-out organoids were then washed with DMEM without phenol red and treated with a solution containing 5 μM fluorescent fatty acid analog C1-BODIPY-C12 (Thermofisher) and 5 μM fatty-acid-free BSA (Sigma-Aldrich) for 30 min at 37°C. Next, the organoids were fixed in 4% paraformaldehyde (VWR) in PBS for 30 min and stained for actin (phalloidin) and cell nuclei (4′,6-diamidino-2-phenylindole; DAPI). Finally, organoids were imaged with a confocal laser scanning microscope (Leica TCS SP8). The intracellular fluorescent signal from C1-BODIPY-C12 was quantified in single confocal z-scans using QuPath 0.3.2.

Kakni P., Jutten B., Teixeira Oliveira Carvalho D., Penders J., Truckenmüller R., Habibovic P, & Giselbrecht S. (2023). Hypoxia-tolerant apical-out intestinal organoids to model host-microbiome interactions. Journal of Tissue Engineering, 14, 20417314221149208.

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Immunophenotyping of Stem Cell Cultures

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In order to confirm the identity of cells isolated and cultured by the aforementioned method, immunofluorescence using a panel of cell markers for SMSCs was performed. Positive and negative markers were selected based on a literature review 18. Cells were plated in four‐chamber slides and allowed to achieve 60% confluence. The cells were then fixed, permeabilized, and blocked with 4% paraformaldehyde (VWR, Radnor, PA), 0.1% Triton X‐100 (Sigma‐Aldrich, St. Louis, MO), and 1% bovine serum albumin (Sigma‐Aldrich). Cells were incubated overnight with primary antibodies against CD31 (Abcam, Cambridge, UK), CD44, CD45, CD90, CD105, CD106, and STRO‐1 (R&D Systems, Minneapolis, MN), followed by incubation with a secondary antibody (Alexa Fluor 488, ThermoFisher Scientific, Waltham, MA). In order to more specifically confirm the identity of these cells, costaining was performed using a similar protocol with antibodies against CD90 (R&D Systems) and CD44 (Abcam). Secondary antibodies for costaining were Alexa Fluor 488 and 594, respectively (ThermoFisher Scientific). All images were obtained using a Keyence BZ‐X700 (Keyence, Osaka, Japan).

Johnson M.B., Niknam‐Bienia S., Soundararajan V., Pang B., Jung E., Gardner D.J., Xu X., Park S.Y., Wang C., Chen X., Baker R.Y., Chen M., Hong Y., Li W, & Wong A.K. (2019). Mesenchymal Stromal Cells Isolated from Irradiated Human Skin Have Diminished Capacity for Proliferation, Differentiation, Colony Formation, and Paracrine Stimulation. Stem Cells Translational Medicine, 8(9), 925-934.

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Characterizing Mesenchymal Cell Lineages

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Gelatin, alizarin red, dimethyl sulfoxide, penicillin−streptomycin, ROCK inhibitor Y27632, scramble-esiRNA, and Twist1-esiRNA were purchased from Sigma-Aldrich (St. Louis, MO). Triton X-100, ethyl acetate, and paraformaldehyde were obtained from VWR (Radnor, USA). Alpha-minimum essential medium (α-MEM), fetal bovine serum (FBS), and goat serum were purchased from Gibco (Carlsbad, CA). Hoechst 33342 was purchased from Pierce Biotechnology (Waltham, MA). The RNeasy Mini Kit was purchased from Qiagen (Hilden, Germany). The SoAdvancedTM Universal SYBR Green Supermix and iScriptTM gDNA Clear cDNA Synthesis Kit were purchased from BioRad (Hercules, CA). Anti-Collagen I (ab34710), Anti-Periostin (ab14041), Anti-Collagen III (ab7778), Anti-Yap1 (ab39361), and Anti-Twist1 antibodies (ab50581) were purchased from Abcam (Cambridge, UK).

Marcus S., Polverino A., Chang E., Robbins D., Cobb M.H, & Wigler M.H. (1995). Shk1, a homolog of the Saccharomyces cerevisiae Ste20 and mammalian p65PAK protein kinases, is a component of a Ras/Cdc42 signaling module in the fission yeast Schizosaccharomyces pombe.. Proceedings of the National Academy of Sciences of the United States of America, 92(13), 6180-6184.

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Immunofluorescent Staining of Primary Neurons

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Primary neurons were fixed with 4% paraformaldehyde (VWR International (Radnor, PA, USA)), washed in DPBS (Thermo Fisher Scientific), then permeabilized using 0.1% Triton X-100 + 1.5% normal donkey serum (Jackson ImmunoResearch, Inc.). Then, the neurons were incubated overnight with primary antibodies and Alexa488- or Cy3-conjugated secondary antibodies for 1 h. Lastly, the slide was layered with a coverslip (Thermo Fisher Scientific) using Fluoromount-G Mounting Medium (Thermo Fisher Scientific).

McKendell A.K., Houser M.C., Mitchell S.P., Wolfe M.S., Berezovska O, & Maesako M. (2022). In-Depth Characterization of Endo-Lysosomal Aβ in Intact Neurons. Biosensors, 12(8), 663.

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Synthesis of Cerium Compound Precursors

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4-ethylphenol (≥97.0%) and CeCl₃·7H₂O (≥99.0%) were purchased from Alfa Aesar (Karlsruhe, Germany). 1,4-phenylenediamine (≥99.0%) and Ce(OOCCH₃)₃·5H₂O (≥99.9%) were purchased from Sigma-Aldrich (Darmstadt, Germany). Ce(NO₃)₃·6H₂O was purchased from Honeywell Fluka (Seelze, Germany). Paraformaldehyde (≥95.0%) and absolute ethanol (≥99.5%) were purchased from VWR (Leuven, Belgium). The starting reagents were used without further purification.

Zhang T., Bonnaud L., Raquez J.M., Poorteman M., Olivier M, & Dubois P. (2020). Cerium Salts: An Efficient Curing Catalyst for Benzoxazine Based Coatings. Polymers, 12(2), 415.

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Preserving ERK1/2 Activity for In Vivo Imaging

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To preserve the intact ERK1/2 activity in vivo for fluorescence microscopy, mice were transcardially perfused before isolation of brain tissue. Thirty minutes prior to transcardiac perfusion, mice were administered with 100/10 mg/kg ketamine/xylazine. Ten minutes prior to perfusion, 20 mg/kg SNC80 (i.p.) or a corresponding volume of saline was administered to the mice. Mice were then perfused with 30 mL of cold PBS and 4% paraformaldehyde (#100503-916, VWR) and were immediately decapitated to collect the brains. The brains were fixated in 4% paraformaldehyde overnight, dehydrated in 30% sucrose, and then embedded in Frozen Section Compound (#3801480, Leica). Frozen brains were sliced at a width of 30 μm using the Leica cryostat and permeabilized in 100% methanol at - 20 °C for 10 minutes. The slices were blocked in 5 % Normal goat serum (#S26-100ml, Millipore Sigma) for an hour then stained with primary antibodies as listed in table S6. For immunofluorescence labeling, the sections were incubated in the secondary antibodies according to the previously established protocol (89 (link)) and as listed in table S6. After the final washing, the nuclei of the sections were stained and the slices were mounted on a glass slide with Vectashield® (#H-1200, Vector lab). Images were acquired with a Nikon confocal microscope and assembled in Adobe Photoshop CS6 (Adobe).

Ko M.J., Chiang T., Mukadam A.A., Mulia G.E., Gutridge A.M., Lin A., Chester J.A, & van Rijn R.M. (2021). β-arrestin–dependent ERK signaling reduces anxiety-like and conditioned fear-related behaviors in mice. Science signaling, 14(694), eaba0245.

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