Transfected cells cultured in 96-well plates (1.5 × 105 cells/well, n = 3, Sigma), were fixed in 4% paraformaldehyde (Applichem GmbH) and permeabilised in PBS-T (0.1% v/v Triton X-100 in PBS) for 5 min. Cells were blocked in PBS-T containing 5% w/v BSA for 10 min and probed with rabbit-anti-NS5 antibodies [17 (link)] in PBS-T, 1% w/v BSA for 1 h at room temperature. After washing with PBS, cells were stained with 1:1000 fluorochrome-conjugated Alexa Fluor 488 (Life technologies, A32790) secondary antibodies in PBS-T, 1% w/v BSA for 1 h at room temperature in the dark. Microplates were analysed using an EVOS microscope (Scale bar: 100 μm). Images were obtained from two independent experiments.
Paraformaldehyde (pfa)
Manufactured by AppliChem
Sourced in Germany
Paraformaldehyde is a solid polymer of formaldehyde. It is used as a fixative and preservative in various laboratory applications.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Tcs sp5 confocal microscope, by Leica (3 mentions)
Potassium ferrocyanide, by Merck Group (3 mentions)
Auriga crossbeam system, by Zeiss (3 mentions)
Sodium cacodylate, by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Alexa fluor 647 conjugated goat anti rabbit, by Thermo Fisher Scientific (2 mentions)
Poly d lysine covered chamber slides, by Corning (2 mentions)
Moviol, by Merck Group (2 mentions)
Dulbecco phosphate buffered saline (dpbs), by Merck Group (1 mentions)
Dapi solution, by Thermo Fisher Scientific (1 mentions)
Fv100, by Olympus (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Phospho histone h2a x ser139 20e3 rabbit mab, by Cell Signaling Technology (1 mentions)
Inverted lsm510 laser scanning microscope, by Zeiss (1 mentions)
Triton x 100, by AppliChem (1 mentions)
Zen 2009 light edition software, by Zeiss (1 mentions)
Toluidine blue o, by Merck Group (1 mentions)
Xylol, by Merck Group (1 mentions)
Entellan neu, by Merck Group (1 mentions)
30 protocols using paraformaldehyde (pfa)
1
Immunofluorescence Assay for DENV NS5 Protein
2
Raman Spectroscopy Characterization of Cells
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For Raman spectroscopy, cells were fixed using 4% paraformaldehyde (AppliChem GmbH, Darmstadt, Germany) in DPBS (Sigma Aldrich LLC). From the suspension, 40 µl was drop cast onto calcium fluoride (CaF2) slides. The slides were then washed in deionised water and the samples were allowed to dry. Raman spectroscopic measurements were performed using a Horiba Jobin Yvon Labram HR800 UV micro-spectroscopy system (Horiba UK Ltd, Middlesex, UK) equipped with a 660 nm solid-state diode laser and calibrated using silicon.
A 100× objective (N.A. 0.95) and a diffraction grating of 300 lines/mm (centred at 1450 cm−1) were used. The laser intensity was set to 100% and the confocal hole was set to 100 μm. A total of 30 spectra were collected from each patient sample. Spectra were recorded with a 20 s integration time and averaged over 3 integrations. Each spectrum was recorded using a 4 × 4 µm raster scan of the centre of each cell.
A 100× objective (N.A. 0.95) and a diffraction grating of 300 lines/mm (centred at 1450 cm−1) were used. The laser intensity was set to 100% and the confocal hole was set to 100 μm. A total of 30 spectra were collected from each patient sample. Spectra were recorded with a 20 s integration time and averaged over 3 integrations. Each spectrum was recorded using a 4 × 4 µm raster scan of the centre of each cell.
3
Quantifying DNA Damage Response
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Cells were transfected with the indicated siRNAs and were trypsinized 24 h after transfection, seeded on glass coverslips in a 24-well plate for 24 h, and irradiated at the indicated doses. γ-H2AX immunofluorescence analysis was carried out as described previously [24 (link)]. At 0 h, 0.5 h, 6 h, 12 h, and 24 h post irradiation, cells were fixed with 4 % paraformaldehyde (15 min, AppliChem, Darmstadt, Germany) at room temperature (RT) and permeabilized by the addition of 0.25 % Triton-X 100 in PBS for 15 min, followed by blocking in 5 % bovine serum albumin (BSA) in PBS for 30 min. Next, cells were incubated with Phospho-Histone H2AX (Ser139) (20E3) rabbit mAb (1:1000; Cell Signal Technology; #9718S), followed by the appropriate Alexa 488-conjugated (green; Molecular Probes) secondary antibodies Subsequently, nuclei were counterstained with DAPI solution (Invitrogen) and coverslips were mounted with Vectashield (Vector Laboratories, Peterborough, UK). Images were taken using an Olympus confocal imaging system (Olympus FV100) for the quantification of γH2AX foci formation. At least three independent experiments were performed for each data point.
4
TRIM25 Intracellular Localization in Colon Cancer
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Staining of intracellular TRIM25 was performed by a confocal microscopy as described [31 (link)]. Colon carcinoma cells were seeded on cover glasses in 12-well plates (neoLab, Heidelberg, Germany) before chemotherapeutic drugs were administered. Thereafter, cells were exposed to 4% paraformaldehyde plus 0.25% Triton X-100 (AppliChem, Darmstadt, Germany) in PBS for 15 min for fixation and permeabilization. After incubation in blocking solution (5% BSA in PBS), a monoclonal anti-TRIM25 antibody was added for 1 h at room temperature. Thereafter, cells were washed several times with PBS before being incubated with a Cy5-conjugated anti-mouse antibody. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies) for 2 min and finally washed with PBS. Stained cells were finally monitored by using an LSM510 inverted laser scanning microscope from Zeiss (Göttingen, Germany). Image analysis was performed with the help of ZEN2009 Light Edition software from Zeiss.
5
Toluidine Blue Staining of Cultured Slices
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Slice cultures were fixed in paraformaldehyde (4%, 0.1 M phosphate buffer; Applichem, Darmstadt, Germany) and rinsed in 0.1 M phosphate-buffered salt solution (PBS). Thereafter, slice cultures were exposed for 20 min to toluidine-blue working solution, which was a mixture of 5 ml stock solution (1 g of Toluidine Blue O in 100 ml of 70% ethanol; Sigma-Aldrich) and 45 ml of 1% NaCl solution (pH 2.0–2.5). Thereafter, 96% ethanol (100 ml of 96% ethanol and 4 drops of acetic acid) was used for color-differentiation of the staining. The differentiation step with strong acid removes unspecific staining of weak acidic structures and, thus, increases the contrast between background and stained cells. The process was stopped using 0.1 M PBS, once the differentiation was clearly visible. After brief rinsing with double distilled water, slice cultures were placed on object plates and dried overnight. The slices were then exposed to xylol (Sigma-Aldrich) for 10 min and embedded with Entellan Neu (Merck Millipore, Schwalbach, Germany).
6
Immunofluorescence Analysis of Protein Markers
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For IF analysis, transfected cells were cultured in 35-mm glass-bottomed dishes (MatTek Corp.), fixed with 4% paraformaldehyde (Applichem GmbH), and permeabilized in PBS-T (0.1% vol/vol Triton X-100 in PBS) for 5 min. Cells were blocked in PBS-T containing 5% wt/vol bovine serum albumin (BSA) for 10 min and probed with primary (Rabbit MAb) antibodies purchased from Cell Signaling Technology (CST) (1:100, cleaved caspase-8 [Asp391, 18C8], cleaved caspase-3 [Asp175, 5A1E], Phospho-Akt [Ser473, D9E] XP, and TIAR [D32D3] XP) or Sigma (1:500, G3BPI: G6046, RRID:AB_1840864) in PBS-T, 1% wt/vol BSA for 1 h at RT. Cells were washed in PBS and stained with 1:500 fluorochrome-conjugated secondary antibodies in PBS-T, 1% wt/vol BSA for 1 h at RT in the dark. Nuclei were counterstained with Hoechst 33342 DNA dye NucBlue Live ReadyProbes (Thermo Fisher) reagent for live cell imaging or 4’,6’-diamidino-2-phenylindole dihydrochloride (DAPI) and SYTO60 fluorescent nucleic acid stain for fixed samples (Invitrogen).
7
Endothelial Barrier Regulation via JAM-A/B
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BrdU-labeled HuMEC-1 were placed on fibronectin-coated 96-well plates and left overnight at 37°C and 5% CO2 to form an endothelial monolayer. HuMEC-1 and PBL were activated with LPS as previously described at 4°C. PBL and HuMEC-1 were co-cultured at 37°C for 120 minutes to simulate a rewarming process [9 (link)]. After removal of the PBL and washing of the HuMEC-1, the endothelial monolayer was fixed with 4% paraformaldehyde (AppliChem), followed by blocking with PBS supplemented with 10% donkey serum (Jackson ImmunoResearch).
Rabbit anti-JAM-A-IgG (1: 200; Santa Cruz Biotechnology) and goat anti-JAM-B-IgG (1: 100; Santa Cruz Biotechnology), donkey anti-rabbit-IgG-PE (1: 100; Santa Cruz Biotechnology) and donkey anti-goat-IgG-FITC (1: 100; Santa Cruz Biotechnology) in PBS containing 1% donkey serum were used for primary and secondary staining of the HuMEC-1, as previously described [9 (link)]. JAM surface protein expression was measured using a microplate reader at 485/535 nm and 530/590 nm, and the fluorescence intensity (FI) was calculated as a ratio to control. Each expression assay was performed in duplicates (PBL from 3 individual volunteers). To reduce cross-contamination of leukocytes, analysis was performed in a separate experiment independently from the transmigration assay.
Rabbit anti-JAM-A-IgG (1: 200; Santa Cruz Biotechnology) and goat anti-JAM-B-IgG (1: 100; Santa Cruz Biotechnology), donkey anti-rabbit-IgG-PE (1: 100; Santa Cruz Biotechnology) and donkey anti-goat-IgG-FITC (1: 100; Santa Cruz Biotechnology) in PBS containing 1% donkey serum were used for primary and secondary staining of the HuMEC-1, as previously described [9 (link)]. JAM surface protein expression was measured using a microplate reader at 485/535 nm and 530/590 nm, and the fluorescence intensity (FI) was calculated as a ratio to control. Each expression assay was performed in duplicates (PBL from 3 individual volunteers). To reduce cross-contamination of leukocytes, analysis was performed in a separate experiment independently from the transmigration assay.
8
Extracellular Nucleotide Detection in Neutrophils
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The extracellular nucleotides were detected using SytoxGreen staining [20 (link)] Briefly, isolated from bone marrow cells neutrophils were transferred to the SytoxGreen dissolved in HBSS (PanEco, Moscow, Russia) 1:1000. Neutrophils were incubated in a black 96-well plate (SPL Life Sciences, Pocheon, Korea) in a concentration of 2 × 105 cells per well at 37 °C and 5% CO2 in the presence of 5 µM of A23187 for 3 h. The samples were analyzed using GloMax Microplate reader Multisystem (Promega, Madison, WI, USA) in a fluorescence mode, filter 490 nm.
Neutrophils were also loaded to the coverglass-bottom slides (Ibidi, Gräfelfing, Germany), incubated 3 h in presence or without of 5 µM of A23187, then fixed with 4% paraformaldehyde (Applichem, Darmstadt, Germany). The samples were stained with anti-histone H3 (citrulline R2 + R8 + R17) antibody (Abcam, Boston, MA, USA, ab5103) and the secondary goat-anti-rabbit PE-conjugated (Sigma-Aldrich, St. Louis, MO, USA, P9537). The nuclei were stained with Hoechst (PanEco, Moscow, Russia), dilution 1:1000.
Neutrophils were also loaded to the coverglass-bottom slides (Ibidi, Gräfelfing, Germany), incubated 3 h in presence or without of 5 µM of A23187, then fixed with 4% paraformaldehyde (Applichem, Darmstadt, Germany). The samples were stained with anti-histone H3 (citrulline R2 + R8 + R17) antibody (Abcam, Boston, MA, USA, ab5103) and the secondary goat-anti-rabbit PE-conjugated (Sigma-Aldrich, St. Louis, MO, USA, P9537). The nuclei were stained with Hoechst (PanEco, Moscow, Russia), dilution 1:1000.
9
Tissue Fixation and Embedding Protocol
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mCC1- (n = 8) or isotype control- (mIgG1; n = 7) treated MP4-immunized mice were sacrificed with CO2 on day 27.6 ± 5.4 after disease onset. After specimen collection for ELISPOT and ELISA all mice were perfused transcardially with 4% paraformaldehyde (PFA) (AppliChem GmbH) in 0.1 M phosphate-buffered saline (PBS), pH 7.4. Draining inguinal lymph nodes and cerebellum were then post-fixed at 4 °C for 24 h. All tissues were embedded in paraffin (Leica TP1020) and stored at 4 °C until analysis.
10
Alcian Blue Staining of Chondrocyte ECM
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Alcian blue staining may detect GAG chains in the ECM of chondrocytes. To prepare Alcian blue solution, 1 g Alcian blue (SERVA Electrophoresis GmbH) was dissolved in 100 ml 3% (v/v) CH3COOH. Human WJ-MSCs and DPSCs at passage 5 were seeded in 24-well cell culture plates (5,000 cells/well) in complete medium, in the presence or in the absence of 1 mg/ml type II collagen. MSCs were cultured for 14 and 24 days in DMEM 2% FBS. Following the treatment period, culture medium was removed, cell monolayers were washed once with PBS 1X and fixed in 4% (w/v) paraformaldehyde (AppliChem GmbH) in PBS 1X for 30 min at room temperature. Cells were washed three times with PBS 1X and stained with Alcian blue staining solution overnight in plate shaker at room temperature. The staining solution was carefully discarded, and wells are washed twice with 3% (v/v) CH3COOH and once with distilled H2O. Images of WJ-MSCs were captured using an inverted OLYMPUS CKX41 microscope equipped with a CMOS color digital camera (SC30) at 10 × magnification. Images of DPSCs were captured using a Nikon DS-Fi3 microscope camera.
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