Polypep

Manufactured by Merck Group

Sourced in Germany

Polypep is a laboratory equipment product designed for the synthesis and analysis of polypeptides. It provides a controlled environment for the step-wise addition of amino acids to form peptide chains.

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Lab products found in correlation

Nitroblue tetrazolium, by Merck Group (5 mentions) Sodium chloride (nacl), by Thermo Fisher Scientific (3 mentions) β nicotinamide adenine dinucleotide, by Merck Group (2 mentions) Sodium chloride (nacl), by Merck Group (2 mentions) Lactic acid, by Merck Group (2 mentions) Paraformaldehyde (pfa), by Merck Group (1 mentions) Sucrose, by Merck Group (1 mentions) Tissue tek oct, by Sakura Finetek (1 mentions) Permount, by Vector Laboratories (1 mentions) Ds ri2 camera, by Nikon (1 mentions) Cytoseal 60, by Thermo Fisher Scientific (1 mentions) Immage nephelometer, by Beckman Coulter (1 mentions) Isopentane, by Thermo Fisher Scientific (1 mentions) Terfenadine, by Merck Group (1 mentions) Dextromethorphan hydrobromide, by Merck Group (1 mentions) Diphenhydramine hydrochloride, by Merck Group (1 mentions) Xylene, by Thermo Fisher Scientific (1 mentions) Acetonitrile, by Thermo Fisher Scientific (1 mentions) Ethanol, by Thermo Fisher Scientific (1 mentions) Methanol, by Thermo Fisher Scientific (1 mentions)

7 protocols using polypep

Cryosectioning and LDH Histochemistry of Frozen Tissues

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Snap-frozen FSEs from -80°C were thawed and fixated in 1% paraformaldehyde (Sigma) for 2 h at 4°C. The FSEs were then put in 20% sucrose (Sigma) in PBS solution overnight at 4°C. FSEs were embedded in Tissue Tek OCT (Sakura Finetek Europe B.V., Alphen aan de Rijn, Netherlands) and sections of 10 µm were cut using a cryotome (Slee MNT, Adamas Instruments B.V., Rhenen, Netherlands). Dried sections were washed in PBS and incubated with LDH solution (2 mM Gly-Gly (Sigma); 0.75% NaCl (Sigma); 5% polypep (Sigma); 1.75 mg/ml β-nicotinamide adenine dinucleotide (Sigma); 3 mg/ml nitroblue tetrazolium (Sigma) in demineralized water of pH 8) for 3 h at 37°C. Sections were washed in tap water at 50°C and in PBS and then stained with Eosin Y for 4 min. Sections were then put in PBS for 1 sec, acetone for 30 sec, acetone/xylene (1:1) for 1 min and xylene for 1 min, before embedding with Eukit Mounting Medium.

Mulder P.P., Vlig M., Elgersma A., Rozemeijer L., Mastenbroek L.S., Middelkoop E., Joosten I., Koenen H.J, & Boekema B.K. (2023). Monocytes and T cells incorporated in full skin equivalents to study innate or adaptive immune reactions after burn injury. Frontiers in Immunology, 14, 1264716.

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Histochemical Assay for Lactate Dehydrogenase

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Frozen sections were left out to air dry for a minimum of 2 hours (range 2–18 hours), then washed twice with PBS for 5 minutes each time. Sections were incubated with freshly prepared LDH solution containing 5% Polypep (Sigma, St. Louis, MO); 2mM Gly-Gly (Sigma, St. Louis, MO ); 0.75% NaCl (Fisher, Hampton, NH); 60mM lactic acid (Dot Scientific Inc., Burton, MI ); 1.75 mg/mL ß-nicotinamide adenine (Sigma, St. Louis, MO); and 3 mg/ml Nitroblue Tetrazolium (Sigma, St. Louis, MO ) pH 8.0, for 3.5 hours at 37°C. Slides were washed twice for 2 minutes each with 50°C tap water, followed by two washes with PBS of 2 minutes each. Tissues were counterstained with aqueous eosin (Newcomer Supply, Middleton, WI) for 4 minutes. Slides were washed with PBS for 1 second, dehydrated with acetone for 30 seconds followed by acetone: xylene (1:1) for 1 minute, and finally with xylene alone for 1 minute. Slides were then cover slipped with Permount (Vector Laboratories, Burlingame, CA).

Karim A.S., Yan A., Ocotl E., Bennett D.D., Wang Z., Kendziorski C, & Gibson A.L. (2018). Discordance between histologic and visual assessment of tissue viability in excised burn wound tissue. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society, 27(2), 150-161.

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LDH Assay in Cryostat Tissue Sections

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An LDH assay^{13 (link)} was performed on cryostat sections after drying at room temperature. Sections were washed twice with PBS for 5 minutes each time. Sections were then incubated with freshly prepared LDH solution^{14 (link)} containing 5% Polypep (Sigma, St. Louis, Missouri), 2 mM Gly-Gly (Sigma), 0.75% NaCl (Fisher, Hampton, New Hampshire), 60 mM lactic acid (Dot Scientific Inc., Burton, Michigan), 1.75 mg/mL ß-nicotinamide adenine (Sigma) and 30 mg Nitroblue Tetrazolium (Sigma) pH 8.0, for 3.5 hours at 37°C. Slides underwent 2 washes with 50°C tap water, followed by 2 washes of PBS for 2 minutes each. Tissues were counterstained with aqueous eosin (Newcomer Supply, Middleton, Wisconsin) for 4 minutes. Slides were washed with PBS for 1 second, dehydrated with acetone for 30 seconds, followed by acetone:xylene (1:1) for 1 minute, and finally dehydrated with xylene alone for 1 minute. Coverslips were placed on the slides with Permount (Vectors, Burlingame, California). Digital images of H&E and LDH-stained slides at ×100 and ×400 were captured on a Nikon DS-Ri2 camera and Nikon Elements BR 4.51.00 software (Nikon, Tokyo, Japan).

Gibson A.L., Bennett D.D, & Taylor L.J. (2017). Improving the histologic characterization of burn depth. Journal of cutaneous pathology, 44(12), 998-1004.

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Assessing Cell Viability via LDH Staining

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Lactate dehydrogenase (LDH) staining was performed on cryosections of ex vivo tissue sections to assess cell viability. Staining was performed as described in Gibson et al.^{27 (link)} Briefly, sections were air-dried for a minimum of 3 hours (range 3–18 hours) then washed twice with PBS for 5 minutes each time. Sections were incubated with freshly prepared LDH solution containing 5% Polypep (Sigma); 2 mM Gly-Gly (Sigma); 0.75% NaCl (Fisher, Hampton, NH); 60 mM lactic acid (Dot Scientific, Burton, MI); 1.75 mg/ml β-nicotinamide adenine (Sigma, St. Louis, MO); and 3 mg/ml Nitroblue Tetrazolium (Sigma) pH 8.0, for 3.5 hours at 37°C. Slides were washed twice for 2 minutes each with 50°C tap water, followed by two washes with PBS for 2 minutes each. Tissues were counterstained with aqueous eosin (Newcomer Supply, Middleton, WI) for 4 minutes. Slides were washed with PBS for 1 second, dehydrated with acetone for 30 seconds followed by acetone:xylene (1:1) for 1 minute, and finally with xylene alone for 1 minute. Slides were then cover slipped with Cytoseal 60 (Thermo Fisher Scientific).

Schroeder A.B., Karim A., Ocotl E., Dones J.M., Chacko J.V., Liu A., Raines R.T., Gibson A.L, & Eliceiri K.W. (2020). Optical imaging of collagen fiber damage to assess thermally injured human skin. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society, 28(6), 848-855.

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Evaluating Cartilage Construct Viability

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Viability of the cartilage constructs was studied by measuring lactate dehydrogenase (LDH) activity, which becomes inactive 24 h after cell death. Cryosections were stained for viability according to a protocol adopted from Stoddart et al. 18 In short, an LDH solution was prepared freshly on the day of testing by adding 60 mM lactic acid (Sigma, L1875) and 1.75 mg/ml b-nicotinamide adenine dinucleotide (Sigma, 43410) to a base solution (40% polypep (Sigma) and 50 mM Gly-Gly (Sigma, G3915)). The pH was adjusted to 8.0 with NaOH and 3 mg/ml Nitroblue Tetrazolium (Sigma, N5514) was added immediately prior to use. After thawing, sections were incubated in reaction medium for 4 h at 37˚C in a humidified chamber protected from light. Sections were then rinsed with warm (50˚C) water and PBS, and fixed with 70% EtOH. Sections were stained with DAPI to visualize cell nuclei.

van Haaften E.E., Ito K, & van Donkelaar C.C. (2017). The initial repair response of articular cartilage after mechanically induced damage. Journal of orthopaedic research : official publication of the Orthopaedic Research Society, 35(6).

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Quantification of Serum and CSF Immunoglobulins

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Serum and CFS IgG, IgM, and albumin were quantified by nephelometry in an Immage nephelometer (Beckman Coulter, Brea, CA). Oligoclonal IgG and IgM bands were studied in paired CSF and serum samples by isoelectric focusing and immunoblotting as described previously (16 (link), 19 (link)). The presence of intrathecal IgG or IgM synthesis was demonstrated by the appearance of two or more oligoclonal bands in CSF, not present in paired serum sample. Lipid specific IgM bands were assessed in the CSF of patients showing intrathecal IgM synthesis as previously described (16 (link)). Briefly, oligoclonal IgM bands are separated by isoelectrofocusing and transferred to nitrocellulose membranes coated with different lipids and blocked with Polypep (Merck). A membrane blocked with Polypep is used as negative control. The presence of anti-lipid IgM antibodies restricted to the CSF is then evidenced by immunoblotting with anti-human IgM antibodies labeled with biotin and with streptavidin labeled with alkaline phosphatase. A representative image of anti-lipid IgM bands is shown in Supplementary Figure 1.

Picón C., Tejeda-Velarde A., Fernández-Velasco J.I., Comabella M., Álvarez-Lafuente R., Quintana E., Sainz de la Maza S., Monreal E., Villarrubia N., Álvarez-Cermeño J.C., Domínguez-Mozo M.I., Ramió-Torrentà L., Rodríguez-Martín E., Roldán E., Aladro Y., Medina S., Espiño M., Masjuan J., Matute-Blanch C., Muñoz-San Martín M., Espejo C., Guaza C., Muriel A., Costa-Frossard L, & Villar L.M. (2021). Identification of the Immunological Changes Appearing in the CSF During the Early Immunosenescence Process Occurring in Multiple Sclerosis. Frontiers in Immunology, 12, 685139.

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Histological and Analytical Techniques

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2,5-dihydroxybenzoic acid (DHB), 9-Aminiacridin hydrochloride (9-AA) trifluoroacetic acid (TFA), paraplast histology grade paraffin, terfenadine, dextromethorphan hydrobromide and diphenhydramine hydrochloride, nitrotetrazolium blue chloride, sodium chloride, 2 mM sodiumhydroxide solution, lactic acid, Nicotinamide adenine dinucleotide hydrate, polypep and Gly-Gly were purchased from Merck (Darmstadt, Germany). M ethanol, water, iso-pentane, ethanol, xylene, isopropanol and acetonitrile (ACN) were obtained from Fisher Scientific (Waltham, MA, USA). Losartan-potassium salt was obtained from Cambridge Bioscience (Cambridge, UK). All solvents used were of analytical grade or higher.

Dannhorn A., Swales J.G., Hamm G., Strittmatter N., Kudo H., Maglennon G., Goodwin R.J, & Takats Z. (2022). Evaluation of Formalin-Fixed and FFPE Tissues for Spatially Resolved Metabolomics and Drug Distribution Studies. Pharmaceuticals, 15(11), 1307.

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