Paraformaldehyde (pfa)

Manufactured by Serva Electrophoresis

Sourced in Germany

Paraformaldehyde is a white, crystalline solid that is commonly used as a fixative in histological and cytological applications. It is a polymer of formaldehyde and is soluble in water and other polar solvents. Paraformaldehyde is typically used to preserve biological samples, such as tissues and cells, by cross-linking proteins and other macromolecules.

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15 protocols using paraformaldehyde (pfa)

Perfusion and Sectioning of CX3CR1 Mice

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CX3CR1 +/GFP mice (n = 6) were transcardially perfused with saline followed by a fixative containing 4% paraformaldehyde (PFA, Serva, Heidelberg, Germany) in phosphatebuffered saline (PBS). Brains were carefully removed and allowed to postfix overnight in the same fixative. Serial sections of 50 µm thickness were prepared using a vibrating microtome (Leica Microsystems, Wetzlar, Germany). Until use, the sections were stored in PBS containing sodium azide at 4 °C.
EAE spinal cord tissue of CX3CR1 CreERT2 :R26-Tomato mice was fixed in 4% PFA (Serva) followed by cryoprotection in 30% sucrose with subsequent freezing and preparation of cryostat sections. Until use, cryostat sections were stored at -20 °C.

Joost E., Jordão M.J.C., Mages B., Prinz M., Bechmann I, & Krueger M. (2019). Microglia contribute to the glia limitans around arteries, capillaries and veins under physiological conditions, in a model of neuroinflammation and in human brain tissue. Brain structure & function, 224(3).

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Cytotoxicity Evaluation of Metal Complexes

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All of the chemicals used throughout experiments (Ni(NO₃)₂, CoCl₂∙6H₂O, CuSO₄∙5H₂O, FeCl₃∙6H₂O, pyridoxal, thiosemicarbazide, Bovine Serum Albumin (BSA)), and the solvents (ethanol, DMSO) were obtained from Merck as p.a. purity chemicals. The ultra-pure water was used for the spectrofluorometric measurements (Milli-Q^® EQ 7000 ultrapure water system). Fetal bovine serum (FBS) and culture mediums RPMI-1640 and Dulbecco’s Modified Eagle Medium (DMEM) were purchased from Capricorn Scientific GmbH (Hessen, Germany). Penicillin Streptomycin solution was purchased from Biological Industries (Cromwell, CT, USA). Trypsin, phosphate-buffered saline (PBS), and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO, USA). 3-(4,5 dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from AppliChem (Maryland Heights, MO, USA). Paraformaldehyde (PFA) was purchased from Serva (Heidelberg, Germany). Crystal violet (CV) was purchased from Biowest (Riverside, MO, USA).

Jevtovic V., Alshamari A.K., Milenković D., Dimitrić Marković J., Marković Z, & Dimić D. (2023). The Effect of Metal Ions (Fe, Co, Ni, and Cu) on the Molecular-Structural, Protein Binding, and Cytotoxic Properties of Metal Pyridoxal-Thiosemicarbazone Complexes. International Journal of Molecular Sciences, 24(15), 11910.

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Lung Tissue Treatment with Heparinase and Pneumolysin

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Fresh human lung tissue explants were obtained from two female patients (52 and 78 years old) suffering from lung carcinoma, who underwent lung resection at local thoracic surgeries. Both patients had a history of chronic obstructive pulmonary disease (patient 1 in early and patient 2 in advanced stage). The study was approved by the ethics committee (Charité-Universitätsmedizin Berlin, Germany, EA2/079/13). Written informed consent was obtained from both patients.
Macroscopically unremarkable (tumor-free) peripheral human lung tissue was dissected into small pieces (1 mm × 1 mm × 3 mm) and incubated for 1 h at 37 °C in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco by Life Technologies, Carlsbad, CA, USA) either with HEP (2 U/400 µl) (heparinase I and III blend from Flavobacterium heparinum, H3917, Sigma-Aldrich, St. Louis, MO, USA) (n = 2 from patient 1, n = 1 from patient 2) or with PLY (0.08 µg/400 µl) (provided by Timothy Mitchell; Mitchell et al. 1989 (link)) (n = 2 from patient 1, n = 1 from patient 2). For comparison with untreated human lung, tissue pieces (n = 2 from patient 1, n = 1 from patient 2) were incubated for 1 h in pure RPMI medium (400 µl). Subsequently, all tissues were fixed and stored in 0.15 M HEPES (Roth, Karlsruhe, Germany) (pH 7.35) containing 1.5% paraformaldehyde and 1.5% glutaraldehyde (both from Serva, Heidelberg, Germany).

Timm S., Lettau M., Hegermann J., Rocha M.L., Weidenfeld S., Fatykhova D., Gutbier B., Nouailles G., Lopez-Rodriguez E., Hocke A., Hippenstiel S., Witzenrath M., Kuebler W.M, & Ochs M. (2023). The unremarkable alveolar epithelial glycocalyx: a thorium dioxide-based electron microscopic comparison after heparinase or pneumolysin treatment. Histochemistry and Cell Biology, 160(2), 83-96.

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Imaging Etiolated Seedling Hypocotyl Anatomy

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Four‐day‐old etiolated seedlings were fixed for 1 h in 4% paraformaldehyde (Serva) in MTSB (50 mm PIPES, 5 nm EGTA, 1 mm MgSO4, pH 6.8) and immobilized in 5% (w/v) water solution of low‐melting agarose (Sigma‐Aldrich). Agarose blocks were mounted onto a Motorized Advance Vibroslice and 100‐μm transversal sections through the hypocotyls were observed with a Zeiss 700 confocal microscope.

Rakusová H., Han H., Valošek P, & Friml J. (2019). Genetic screen for factors mediating PIN polarization in gravistimulated Arabidopsis thaliana hypocotyls. The Plant Journal, 98(6), 1048-1059.

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Fabrication and Characterization of PLGA Nanoparticles

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Poly(D,L-lactide-co-glycolide) (PLGA polymer with carboxylic terminal group, 50/50 of inherent viscosity midpoint 0.2 dL/g; M_W 10,000–18,000) was purchased from LACTEL Absorbance Polymers (Birmingham, AL, USA). D-mannitol and polyvinyl alcohol (PVA, MW 30,000–70,000) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chloroform was purchased from Ruskhim (Moscow, Russia). Dimethyl sulphoxide (DMSO), Tween 80, and phosphate buffered saline (PBS) were purchased from Amreso (Solon, OH, USA). Trypsin and fetal bovine serum (FBS) were purchased from Hyclone (Logan, UT, USA). Ethylenediaminetetraacetic acid (EDTA), 2′-7′dichlorofluorescin diacetate (DCFH-DA), and paraformaldehyde were purchased from Serva (Heidelberg, Germany). DMEM and RPMI 1640 culture medium were purchased from Gibco (Waltham, MA, USA). 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyltetrazoliumbromide (MTT), giemsa, mowiol, and o-phthaldialdehyde were purchased from Sigma-Aldrich (St. Louis, MO, USA). MitoSox Red was purchased from Invitrogen (Waltham, MA, USA). MitoStep was purchased from Immunostep (Salamanca, Spain). Annexin V-FITC/PI kit was purchased from Biolegend (San. Diego, CA, USA). TUNEL and Lipid Peroxidation (MDA) Colorimetric assay kits were purchased from Biovision (Milpitas, CA, USA). All other chemicals were used as HPLC grade or extra pure grade.

Mollaeva M.R., Nikolskaya E., Beganovskaya V., Sokol M., Chirkina M., Obydennyi S., Belykh D., Startseva O., Mollaev M.D, & Yabbarov N. (2021). Oxidative Damage Induced by Phototoxic Pheophorbide a 17-Diethylene Glycol Ester Encapsulated in PLGA Nanoparticles. Antioxidants, 10(12), 1985.

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Isolated Cardiac Muscle Experiments

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For the present study, the compound, berberine chloride hydrate (97% high-performance liquid chromatography), was purchased from Alfa Aesar, Johnson Matthey Co., Heysham, England. Glutaraldehyde 25% electron-microscopy grade was purchased from SERVA Electrophoresis GmbH, Heidelberg, Germany. Paraformaldehyde (catalog number UN2213) was purchased from SERVA Electrophoresis GmbH. Metaprolol was purchased from AstraZeneca Pharma Ltd., Bangalore, India. Propranolol was purchased from Cipla, Mumbai, India. Isoprenaline was purchased from SGPharma, Mumbai, India. Adrenaline was purchased from Neon Laboratories Ltd., Mumbai, India. Potassium chloride and sodium chloride were purchased from Loba Chemie Pvt. Ltd. (Mumbai, India). Sodium bicarbonate was purchased from RFCL (RANKEM) Ltd., New Delhi, India, and calcium chloride from Fisher Scientific, Mumbai, India. Other reagents were of analytical grade.

Ali S.A, & Naaz I. (2015). Understanding the ultrastructural aspects of berberine-induced skin-darkening activity in the toad, Bufo melanostictus, melanophores. Journal of Microscopy and Ultrastructure, 3(4), 210-219.

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Immunostaining of LC3-II in Cells

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To examine the intensity and the pattern of LC3-II immunostaining, the cells were seeded on glass cover slides in 12-well plates and left to attach overnight. After the treatment with fisetin and/or paclitaxel, the cells were fixed with 4 % paraformaldehyde (Serva; Heidelberg, Germany) in PBS for 30 min, washed three times with PBS, permeabilized with 0.25 % Triton X-100 (Serva; Heidelberg, Germany) in PBS for 5 min and blocked with 1 % BSA (Sigma-Aldrich; St. Louis, MO, USA) in PBS (BSA–PBS pH 7.6) for 20 min. The staining of LC3-II was performed using the rabbit anti-LC3-II antibody (Thermo Scientific; Rockford, USA) diluted 1:2000 in 1 % BSA–PBS (1 h, RT, a moist chamber). After rinsing three times with 1 % BSA–PBS, the cells were incubated with Alexa Fluor 488-labeled goat anti-rabbit secondary antibody (Life Technologies Corp.; Carlsbad, CA, USA) diluted 1:100 in PBS (60 min, RT, a moist chamber in the dark). Following three washing steps with PBS, the cell nuclei were counterstained with DAPI (diluted 1:20,000 in distilled water; Sigma-Aldrich; St. Louis, MO, USA) for 10 min. Finally, the slides were rinsed three times with distilled water, mounted with Aqua-Poly/Mount (Polysciences; Warrington, PA) and examined using Nikon Eclipse E800 fluorescence microscope and NIS-Elements 4.0 software (all from Nikon; Tokyo, Japan).

Klimaszewska-Wisniewska A., Halas-Wisniewska M., Tadrowski T., Gagat M., Grzanka D, & Grzanka A. (2016). Paclitaxel and the dietary flavonoid fisetin: a synergistic combination that induces mitotic catastrophe and autophagic cell death in A549 non-small cell lung cancer cells. Cancer Cell International, 16, 10.

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Colony Formation Assay for Evaluating Anti-tumor Compounds

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Cells were seeded in 6-well plates and incubated with dimethyl sulfoxide (DMSO), izTRAIL, Dinacicllib, or a combination of the latter for 24 h. After incubation for 7 days, cells were fixed in 4% Paraformaldehyde (PFA) (Serva, Heidelberg, Germany) for 15 min at room temperature, and stained with crystal violet (Sigma-Aldrich^®, St. Louis, MI, USA; 250 mg crystal violet powder in 250 mL 95% methanol)
Colony formation assay was performed as described previously [27 ] according to the protocol of Millipore (Catalog No. ECM570, Merck KGaA, Darmstadt). Respective treatments were added to the top agar. The cells were incubated for 28 days until colonies were formed. Formed colonies were photographed for three times in a bright field at specific positions located on each fan-shaped center of every well divided into three equal parts and subsequentially visualized and quantified using MTT staining. The colony count was performed using ImageJ (National Institute of Health, Bethesda, MD, USA).

Shen X., Kretz A.L., Schneider S., Knippschild U., Henne-Bruns D., Kornmann M., Lemke J, & Traub B. (2023). Evaluation of CDK9 Inhibition by Dinaciclib in Combination with Apoptosis Modulating izTRAIL for the Treatment of Colorectal Cancer. Biomedicines, 11(3), 928.

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Artificial Infection of Honeybee Midguts

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For experiments on artificial infection A. mellifera carnica brood was obtained from a private housekeeper in Belarus. The absence of natural microsporidia infection was confirmed by selective examination using microscopy and PCR. Foraging bees were planted in 0.5 l plastic bottles and then fed sugar solution supplemented with V. ceranae spores. A standard infection doze was one million spores per bee.
Ten days post-infection bees were dissected to isolate midguts for IFA and immunoblotting. For immunoblotting, midguts were homogenated in TB and heated at 95 °C for 10 min with an equal volume of 2× sample buffer (125 mM Tris-Cl (pH 6.8), 4% SDS, 10% 2-mercaptoethanol, 20% glycerol). For IFA the midguts were fixed in PBS (phosphatebuffered saline) with 4% paraformaldehyde (Serva, Germany) for 24 h, washed in PBS, incubated in 30% sucrose for cryoprotection, and frozen in liquid nitrogen. Frozen sections (10 µm thickness) were prepared with the Microm HM 520 cryotome and placed on microscope slides. Immunoblotting and IFA with newly raised antibodies were performed as described before (Senderskiy et al., 2014b; Timofeev et al., 2017) in three independent infection experiments.

Senderskiy I.V., Ignatieva A., Kireeva D.S., & Vv D. (2021). Production of polyclonal anti-β-tubulin antibodies and immunodetection of Vairimorpha (Nosema) ceranae (Opisthosporidia: Microsporidia) proliferative stages in the midguts of Apis mellifera and in the Sf9 cell culture. Protistology, 15(1).

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In vitro Nosema ceranae Spore Infection of Sf9 Cells

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Sf9 cell line derived from fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae) pupal ovarian tissue, was obtained from ECACC General collection (ECACC 89070101). Cells were cultured in Sf900III SFM (Thermo Fisher Scientific, MA) and maintained according to manufacturer's inst-Igor V. Senderskiy, Anastasiya N. Ignatieva, Darya S. Kireeva and Viacheslav V. Dolgikh ructions. Cells for infections were in the mid-log phase growth and their viability was over 90%.
V. ceranae spores for cell culture infections were obtained from artificially infected living bees. After midgut dissection and homogenization in distilled water, spores were additionally purified on 50% Percoll gradient and sterilized with antiseptic Multicide for 30 min followed by washing in sterile water. Then 10 8 spores in water were dried in a well of 6-well plate for 30 min and cell suspension has been added at a ratio of 1 cell per 200 spores. In 5-day post-infection the cells were separated from spores using 20% Percoll gradient.
The cells were fixed for 24 h by the addition of 8% paraformaldehyde (Serva, Germany) prepared in PBS to an equal volume of cell suspension. IFA with polyclonal antibodies was carried out as previously described (Timofeev et al., 2017) . The experiments on in vitro infection followed by fixation and IFA were independently repeated 3 times.

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