After rinsing with PBS, cells were fixed in 10% wt/vol. formaldehyde solution (AppliChem, Darmstadt, Germany) for 30 minutes, rinsed in dH2O and dehydrated in 60% vol./vol. isopropanol for 4 min. Subsequently, cells were stained with 0.3% wt/vol. Oil-Red-O (O0625; Sigma-Aldrich, Buchs, Switzerland) dissolved in 100% isopropanol that was freshly diluted in dH2O to 60% vol./vol. After 5 minutes, cells were rinsed three times with tap water and imaged using an Axio Observer microscope platform (Zeiss, Jena, Germany). Oil Red O was extracted by adding 100% isopropanol for 30 minutes while shaking the plate, and optical density (OD) was measured at 520 nm.
Formaldehyde solution
Manufactured by AppliChem
Sourced in Germany
4% Formaldehyde solution is a laboratory reagent used for fixation and preservation of biological samples. It is a clear, colorless liquid with a characteristic odor. The solution contains 4% formaldehyde in water.
Automatically generated - may contain errors
Lab products found in correlation
Poly l lysine hydrobromide, by Merck Group (2 mentions)
Anti v5 tag primary antibody, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (2 mentions)
Zen black 2, by Zeiss (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Prolong gold, by Thermo Fisher Scientific (2 mentions)
High precision microscope cover glasses, by Marienfeld (2 mentions)
Axio observer microscope, by Zeiss (1 mentions)
Oil red o, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Edta monovette, by Sarstedt (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
5 protocols using formaldehyde solution
1
Oil Red O Staining and Quantification
2
B Cell Activation and Signaling
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PBMC or isolated CD19+ B cells from healthy volunteers were stimulated with anti-µ-F(ab’)2 (5 µg/ml, Dianova, Germany). Some cells were incubated with ADO (1 mM) (Sigma-Aldrich, USA) for two hours or surface-stained with CD73 eFluor450 before stimulation. The stimulation was stopped by adding 10% formaldehyde solution (AppliChem, Germany). Cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, USA) kept in 50% methanol at − 20 °C for 10 min. Cells were washed and stained with p-BTK (pY223) PE for 45 min. at room temperature before FACS analysis.
3
Confocal Imaging of V5-tagged 293T Cells
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For the confocal imaging of 293T cells, high precision microscope cover glasses (Marienfeld) were coated with poly-L-lysine hydrobromide (p6282, Sigma-Aldrich) according to the manufacturer’s protocol. Cells were seeded onto cover glasses in normal growth medium and fixed in 4% Formaldehyde solution (AppliChem) in PBS 1x after 24h of incubation. Permeabilization and blocking of samples was performed in blocking solution (10% FCS, 0.3% Saponin (47036, Sigma-Aldrich) in PBS 1x) for 1h rocking. Anti-V5 Tag primary antibody (Thermo Fischer Scientific, #46-0705) was diluted 1:500 in blocking solution and applied for 2h at room temperature, rocking. Samples were washed three times in blocking solution and anti-mouse Alexa Fluor 594 (Thermo Fischer Scientific, #A-11005) was applied 1:400 in blocking solution for 1h at room temperature, rocking. After three times washing in blocking solution nuclei were counterstained with DAPI 1:1000 in PBS 1x, for 10min, rocking. Cover glasses were mounted onto microscopy slides using ProLong Gold (Thermo Fischer Scientific) antifade mountant. Image acquisition was performed on a confocal laser scanning microscope (Zeiss LSM 780, Carl Zeiss AG), equipped with an Airyscan detector using ZEN black 2.3 (Carl Zeiss AG).
4
Confocal Imaging of V5-tagged 293T Cells
5
Tissue Sampling and Storage Protocol
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The rats were humanely killed in deep anaesthesia either 24 h or 5 d after TxT or sham procedure by exsanguination plus setting a bilateral pneumothorax. Blood was collected in ethylenediaminetetraacetic acid (EDTA)-Monovette (Sarstedt). Blood samples were centrifuged at 340g for 10 min and plasma was stored at -70°C. The pulmonary circulation was flushed with 10 mL PBS. The lower right lung lobe and the left lung were clamped while the rest of the lung was flushed with 2.5 mL cold PBS by cannulated trachea. It was mixed with 50 µL protease inhibitor cocktail (Sigma-Aldrich) and was used for cell counting and cytokine measurements. After that, lung was flushed two times with 5 mL PBS for flow cytometry analysis. The left lung was flushed with 4% formaldehyde solution (AppliChem) and stored. The lower lung lobe, liver, spleen, kidney, brain and gonads were frozen in liquid nitrogen for Alu polymerase chain reaction (PCR) analysis.
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