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Paraformaldehyde pbs

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Paraformaldehyde/PBS is a laboratory reagent used for fixation and preservation of biological samples. It is a solution of paraformaldehyde, a polymer of formaldehyde, in phosphate-buffered saline (PBS). This solution helps maintain the structure and morphology of cells, tissues, or other biological specimens during various experimental procedures.

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Lab products found in correlation

Lsm 780 confocal microscope, by Zeiss (6 mentions) Triton x 100, by Merck Group (4 mentions) Triton x 100 pbs, by Thermo Fisher Scientific (3 mentions) Prolong gold antifade reagent, by Thermo Fisher Scientific (3 mentions) Axio observer z1, by Zeiss (3 mentions) Hoechst 33342, by Thermo Fisher Scientific (3 mentions) Dimethyl sulfoxide (dmso), by Merck Group (2 mentions) Infinite m1000 microplate reader, by Tecan (2 mentions) Direct red 80, by Merck Group (2 mentions) Primary anti prlr, by Santa Cruz Biotechnology (2 mentions) Plan apochromat x63 1.2 water dic objective, by Zeiss (2 mentions) Alexa fluor 488, by Thermo Fisher Scientific (2 mentions) Paraformaldehyde (pfa), by Merck Group (2 mentions) Rabbit anti human intelectin 94 145 antibody, by Phoenix Pharmaceuticals (2 mentions) Isotype matched control rabbit igg, by Abmart (2 mentions) Dab kit, by Zhongshan Golden Bridge Biotechnology (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Bsa pbs, by Merck Group (2 mentions) Lsm 700 confocal microscope, by Zeiss (2 mentions) Lsm 510 meta, by Zeiss (2 mentions)

41 protocols using paraformaldehyde pbs

1

Histological Analysis of Endobronchial and Lung Tissues

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We stained 2 μm sections with hematoxylin and eosin (H&E) or rabbit-anti-human intelectin (94–145) antibody (1:400; Phoenix Pharmaceuticals, US) or an isotype matched control rabbit IgG (1:2000; Abmart Shanghai, China) for endobronchial and skin biopsies. For endobronchial biopsies, observers who were blinded to the clinical status of the subjects counted numbers of eosinophils/mm2 submucosa, and calculated basement membrane thickness (BMT). We used the mean of 50 measurements taken over a distance of at least 1 mm to calculate BMT as previously described 57 (link).
Mouse lungs were fixed for 5 min by instillation of 4% paraformaldehyde-PBS (Sigma, USA) through a tracheal catheter at a transpulmonary pressure of 15 cmH2O and lungs were fixed in 4% paraformaldehyde-PBS overnight. Five-μm-thick sections were used for immunohistochemistry with rabbit-anti-human intelectin (94–145) antibody (1:400; Phoenix Pharmaceuticals, USA) or an isotype matched control rabbit IgG (1:2000; Abmart Shanghai, China). Antibodies were detected using the DAB kit (Zhongshan Goldenbridge Biotechnology, China) as directed by the manufacturer. Lung sections were also stained with Periodic acid-Schiff (PAS; Goodbio technology, China) staining for detection of mucus. The number of PAS-staining-positive cells was counted in four random fields for each lung section at ×200 magnification 58 (link).
Yi L., Cheng D., Zhang K., Huo X., Mo Y., Shi H., Di H., Zou Y., Zhang H., Zhao J., Xu Y., Erle D.J, & Zhen G. (2017). Intelectin contributes to allergen-induced IL-25, IL-33 and TSLP expression and type 2 response in asthma and atopic dermatitis. Mucosal immunology, 10(6), 1491-1503.
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2

Histological Analysis of Endobronchial and Lung Tissues

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We stained 2 μm sections with hematoxylin and eosin (H&E) or rabbit-anti-human intelectin (94–145) antibody (1:400; Phoenix Pharmaceuticals, US) or an isotype matched control rabbit IgG (1:2000; Abmart Shanghai, China) for endobronchial and skin biopsies. For endobronchial biopsies, observers who were blinded to the clinical status of the subjects counted numbers of eosinophils/mm2 submucosa, and calculated basement membrane thickness (BMT). We used the mean of 50 measurements taken over a distance of at least 1 mm to calculate BMT as previously described 57 (link).
Mouse lungs were fixed for 5 min by instillation of 4% paraformaldehyde-PBS (Sigma, USA) through a tracheal catheter at a transpulmonary pressure of 15 cmH2O and lungs were fixed in 4% paraformaldehyde-PBS overnight. Five-μm-thick sections were used for immunohistochemistry with rabbit-anti-human intelectin (94–145) antibody (1:400; Phoenix Pharmaceuticals, USA) or an isotype matched control rabbit IgG (1:2000; Abmart Shanghai, China). Antibodies were detected using the DAB kit (Zhongshan Goldenbridge Biotechnology, China) as directed by the manufacturer. Lung sections were also stained with Periodic acid-Schiff (PAS; Goodbio technology, China) staining for detection of mucus. The number of PAS-staining-positive cells was counted in four random fields for each lung section at ×200 magnification 58 (link).
Yi L., Cheng D., Zhang K., Huo X., Mo Y., Shi H., Di H., Zou Y., Zhang H., Zhao J., Xu Y., Erle D.J, & Zhen G. (2017). Intelectin contributes to allergen-induced IL-25, IL-33 and TSLP expression and type 2 response in asthma and atopic dermatitis. Mucosal immunology, 10(6), 1491-1503.
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3

Immunofluorescence Staining of Extracellular Matrix

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BM- and AD-ECM were rehydrated by incubation in PBS for 1 h at 37 °C and then treated with DNase (100 units/mL; Sigma-Aldrich) for 1 h at 37 °C. After removal of the enzyme and a brief rinse in PBS, the ECMs were fixed with 4% paraformaldehyde/PBS (Sigma-Aldrich) for 30 min at room temperature. After fixation, the ECMs were washed three times with PBS and then stored in PBS until commencing the staining procedure.
For IF staining, ECMs were first treated with blocking solution (Bloxall, Vector Labs, Inc., Burlingame, CA) for 30 min at room temperature and then incubated for 1 h at 4 °C with primary antibody specific for human type VI collagen (1:200 dilution; polyclonal rabbit IgG; EMD Millipore, Temecula, CA). Non-specific rabbit IgG (Sigma-Aldrich) was employed as a negative control (1:200). Unbound primary antibody was removed by washing with cold (4 °C) PBS. After washing, the ECMs were incubated with blocking solution again for 10 min at room temperature. Secondary antibody (1:200; FITC-conjugated goat anti-rabbit IgG, Sigma-Aldrich) was then added and the incubation continued for 1 h at 4 °C. Florescence microscopy was performed at 10 × magnification using an Olympus IX73 inverted microscope.
Marinkovic M., Block T.J., Rakian R., Li Q., Wang E., Reilly M.A., Dean D.D, & Chen X.D. (2016). One size does not fit all: developing a cell-specific niche for in vitro study of cell behavior. Matrix biology : journal of the International Society for Matrix Biology, 52-54, 426-441.
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4

TUNEL Assay for DNA Fragmentation Detection

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To detect DNA fragmentation, TUNEL assay was performed 5 h after the treatments. Cells grown on coverslips were fixed in 4% paraformaldehyde/PBS (Sigma-Aldrich) for 15 min at 4 °C, washed thrice with PBS, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 2 min and incubated with TUNEL reaction mixture (Roche, Indianapolis, IN, USA) for 1 h at 37 °C. Cells were washed with PBS, mounted in ProLong Gold and observed under a Confocal Laser Scanning Microscope (CLSM, Olympus XT7).
Soriano J., Mora-Espí I., Alea-Reyes M.E., Pérez-García L., Barrios L., Ibáñez E, & Nogués C. (2017). Cell Death Mechanisms in Tumoral and Non-Tumoral Human Cell Lines Triggered by Photodynamic Treatments: Apoptosis, Necrosis and Parthanatos. Scientific Reports, 7, 41340.
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5

Quantifying Mitochondrial Superoxide Levels

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•-rate Superoxide anion levels were quantified using the MitoSOX™ Red Mitochondrial Superoxide Indicator dye (Life Technologies). MitoSOX reagent working solution (5 μM) was prepared by diluting MitoSOX reagent stock solution (5 mM in DMSO (Sigma Aldrich)) in Neurobasal A without phenol red buffer. At the end of light exposure, medium was replaced with 350 μl of MitoSOX reagent working solution; the wellplate was then incubated for 10 min at 37 °C in the dark. Cells were further carefully washed 3 times with warm PBS; 350 μl of fresh PBS was added to wells. The fluorescent signal was read on an Infinite M1000 microplate reader (Tecan, Männedorf, Switzerland): λ↑ = 510 nm, λ↓ = 580 nm. Measured values were normalized according to control cells (in the dark) in normal conditions considered as 1 and also according to viability. Cells were then fixed with 4% paraformaldehyde-PBS (Sigma-Aldrich) for further immunostaining and microscopic imaging.
Marek V., Potey A., Réaux-Le-Goazigo A., Reboussin E., Charbonnier A., Villette T., Baudouin C., Rostène W., Denoyer A, & Mélik Parsadaniantz S. (2019). Blue light exposure in vitro causes toxicity to trigeminal neurons and glia through increased superoxide and hydrogen peroxide generation. Free radical biology & medicine, 131.
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6

Mitochondrial Activity Imaging Assay

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Mitochondria status was assessed with MitoTracker™ Deep Red FM dye (Life Technologies, Carlsbad, CA, USA). MitoTracker reagent working solution (25 μM) was prepared by diluting MitoSOX reagent stock solution (1 mM in DMSO (Sigma Aldrich, St. Louis, MO, USA)) in Neurobasal w/o phenol red buffer. At the end of light exposure, medium was replaced with 350 μl of MitoTracker reagent working solution; the well-plate was then incubated for 30 min at RT in the dark. Cells were then fixed with 4% paraformaldehyde-PBS (Sigma-Aldrich) and counterstained with DAPI, for further microscopic imaging.
Marek V., Potey A., Réaux-Le-Goazigo A., Reboussin E., Charbonnier A., Villette T., Baudouin C., Rostène W., Denoyer A, & Mélik Parsadaniantz S. (2019). Blue light exposure in vitro causes toxicity to trigeminal neurons and glia through increased superoxide and hydrogen peroxide generation. Free radical biology & medicine, 131.
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7

Histological and Immunofluorescence Analysis of Cardiac Tissue

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Histology and immunofluorescence staining were performed as described58 (link). Hearts were excised, rinsed in cold PBS and fixed in 4% paraformaldehyde/PBS (Sigma Aldrich) for 24–48 hours. The tissue was subsequently dehydrated through an increasing ethanol series, cleared in toluol and embedded in paraffin. 5 µm paraffin sections were stained with hematoxylin & eosin (Carl Roth) to assess overall cardiac morphology or with Sirius Red (Direct Red 80, Sigma Aldrich) to visualize myocardial fibrosis.
For immunofluorescence staining paraffin sections were deparaffinized, rehydrated and heat mediated antigen retrieval was performed in sodium citrate buffer (10 mM, pH 6.0) for 20 minutes. After blocking in antibody solution containing 5% normal goat serum (Jackson ImmunoResearch) for 1 hour, sections were incubated over night with primary antibodies at 4 °C. Secondary antibody detection was performed at room temperature for 1 hour using Alexa Fluor 488 or 555 conjugated secondary antibodies (Life Technologies, 1:500). Nuclei were stained with DAPI or TO-PRO-3 (Life Technologies) and sections were mounted in ProLong Gold antifade reagent (Life Technologies).
Hennig M., Ewering L., Pyschny S., Shimoyama S., Olecka M., Ewald D., Magarin M., Uebing A., Thierfelder L., Jux C, & Drenckhahn J.D. (2019). Dietary protein restriction throughout intrauterine and postnatal life results in potentially beneficial myocardial tissue remodeling in the adult mouse heart. Scientific Reports, 9, 15126.
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8

Organ Preparation and Histological Analysis

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Hearts, kidneys, and lungs from neonatal mice were prepared on postnatal day 1, and hearts, livers, kidneys, and spleens from adult mice were prepared at the age of 11 weeks. Adult mice were euthanized by cervical dislocation, and neonates were decapitated. Organs were excised, rinsed in cold PBS, weighed, snap‐frozen in liquid nitrogen, and stored at −80°C. If organs were used for histological analyses, they were fixed in 4% paraformaldehyde/PBS (Sigma) for 24 to 48 hours. The tissue was subsequently dehydrated through an increasing ethanol series, cleared in toluol, and embedded in paraffin. Five‐micrometer paraffin sections were stained with hematoxylin and eosin (Carl Roth, Karlsruhe, Germany) to assess overall cardiac morphology and tissue composition or with Sirius Red (Direct Red 80, Sigma) to visualize myocardial fibrosis.
Hennig M., Fiedler S., Jux C., Thierfelder L, & Drenckhahn J. (2017). Prenatal Mechanistic Target of Rapamycin Complex 1 (m TORC1) Inhibition by Rapamycin Treatment of Pregnant Mice Causes Intrauterine Growth Restriction and Alters Postnatal Cardiac Growth, Morphology, and Function. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 6(8), e005506.
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9

Confocal Imaging of Prolactin Receptor Variants

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Confocal imaging was performed as previously described (Gorvin et al. 2018b (link)). Cells were plated in six-well plates containing poly-l-lysine-treated coverslips and cultured at 37°C. Cells were transiently transfected with 1000 ng of either WT or variant PRLR expression constructs. After 24 h, cells were fixed in 4% paraformaldehyde/PBS (Sigma-Aldrich), permeabilised in 1% Triton-X100/PBS (Thermo Scientific), and immunostained with primary anti-PRLR (1:200, Santa Cruz Biotechnology) and secondary antibody Alexa Fluor-488 (1:1000, Molecular Probes). Cells were mounted in Prolong Gold Antifade reagent (Invitrogen). Images were captured using a Zeiss LSM780 confocal microscope with a Plan-Apochromat x63/1.2/water DIC objective. An argon laser (488 nm) was used to excite Alexa Fluor-488.
Gorvin C.M., Newey P.J, & Thakker R.V. (2022). Identification of prolactin receptor variants with diverse effects on receptor signalling. Journal of Molecular Endocrinology, 70(3), e220164.
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10

Cytotoxic NK Cell-Tumor Cell Interaction

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The NK cells and tumor cells were stained with CellTracker Green 5-chloromethylfluoresceindiacetate (CMFDA) and Orange [5-(and-6)-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine] (CMTMR) (Thermo Fisher Scientific, Inc.) for 30 min at 37°C. On the following day, the cells were co-cultured for 45 min, fixed for at least 30 min in 4% (w/v) paraformaldehyde/PBS (Sigma-Aldrich; Merck KGaA) at 4°C and analyzed on FACScalibur (BD Biosciences) or AccuriC6 (BD Biosciences) and the data were entered into the respective CellQuest (version 4.0) or C6 Flow (version 227.4) software (both from BD Biosciences). Double-stained events, corresponding to the interaction between a tumor cell and at least one NK cell, were compared to the whole stained tumor cell population.
Carré T., Thiery J., Janji B., Terry S., Gros G., Meurice G., Kieda C., Olive D, & Chouaib S. (2020). Selection of tumor-resistant variants following sustained natural killer cell-mediated immune stress. Oncology Reports, 45(2), 582-594.
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