Vero.E6 cells were seeded in a 96-well cell culture plate (Corning; 3340) 1 day prior to the assay, at a density of 20,000 cells per well. Mouse serum samples were heat inactivated at 56°C for 1 h prior to use. Serum dilutions were prepared in 1× minimal essential medium (MEM; Gibco) supplemented with 1% FBS. Virus was diluted to 10,000 50% tissue culture infectious doses (TCID50s)/mL, and 80 μL of virus and 80 μL of serum were incubated together for 1 h at RT. After the incubation, 120 μL of virus–serum mixture was used to infect cells for 1 h at 37°C. Next, the virus–serum mix was removed and 100 μL of each corresponding dilution was added to each well. A volume of 100 μL of 1× MEM were also added to the plates to get to a total volume of 200 μL in each well. Cells were incubated at 37°C for 3 days and later fixed with 10% paraformaldehyde (Polysciences) for 24 h. After 24 h, the paraformaldehyde solution was discarded and cells were permeabilized for intracellular staining. Staining and quantification were performed as previously described (40 , 44 (link), 54 (link), 55 (link)).
Paraformaldehyde (pfa)
Manufactured by Polysciences
Sourced in United States, Germany, United Kingdom, Panama
Paraformaldehyde is a solid polymer of formaldehyde. It is commonly used as a fixative in histology and cell biology applications to preserve the structure and morphology of cells and tissues.
Automatically generated - may contain errors
Lab products found in correlation
1200 ex transmission electron microscope, by JEOL (26 mentions)
Glutaraldehyde, by Polysciences (24 mentions)
Eponate 12 resin, by Ted Pella (19 mentions)
Osmium tetroxide, by Polysciences (19 mentions)
Aqueous uranyl acetate, by Ted Pella (18 mentions)
Amt 8 megapixel digital camera, by Advanced Microscopy Techniques (13 mentions)
Ultracut uct ultramicrotome, by Leica (13 mentions)
Amt image capture engine v602, by Advanced Microscopy Techniques (12 mentions)
Uranyl acetate, by JEOL (9 mentions)
710 confocal microscope, by Zeiss (7 mentions)
Ultracut uct7 cryo ultramicrotome, by Leica (5 mentions)
Triton x 100, by Merck Group (5 mentions)
Bovine serum albumin (bsa), by Merck Group (5 mentions)
Ultracut uc7 ultramicrotome, by Leica (5 mentions)
Rabbit anti ha antibody, by Merck Group (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Lsm 880 confocal microscope, by Zeiss (4 mentions)
Axio imager a1 microscope, by Zeiss (4 mentions)
Oil red o, by Merck Group (3 mentions)
Fibronectin, by Merck Group (3 mentions)
143 protocols using paraformaldehyde (pfa)
1
SARS-CoV-2 Neutralization Assay in Vero.E6 Cells
2
Fixation and Sectioning of Airway Epithelium
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At 4 days postinfection (DPI), 200 μl of 1x PBS was added to the apical site of the Transwell for washing before PFA fixation. In the basal side of the Transwell inserts was 200 μl of 1x PBS. For PFA fixation, 200 μl of 4% PFA (Polysciences) was added to the upper chamber of the Transwells and incubated for 30 min, and the Transwells were further maintained overnight in 4% PFA prior to removal from high biocontainment. The PFA fixation protocol was approved as an inactivation method for coronaviruses by the RML Institutional Biosafety Committee. The PFA-fixed airway epithelium was paraffin-embedded and sectioned at a thickness of 5 μm for slide preparation as previously described [43 (link)].
3
Mitochondrial Ultrastructural Analysis
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Samples were fixed in 2% paraformaldehyde/2.5% glutaraldehyde (Polysciences) in 100 mM sodium cacodylate buffer, pH 7.2 for 1 hr at room temperature. Samples were washed in sodium cacodylate buffer and postfixed in 1% osmium tetroxide (Polysciences) for 1 hr. Next, samples were rinsed in dH2O prior to en bloc staining with 1% aqueous uranyl acetate (Ted Pella) for 1 hr. Following several rinses in dH2O, samples were dehydrated in a graded series of ethanol and embedded in Eponate 12 resin (Ted Pella). Sections of 95 nm were cut with a Leica Ultracut UCT ultramicrotome (Leica Microsystems), stained with uranyl acetate and lead citrate, and viewed on a JEOL 1200 EX transmission electron microscope (JEOL USA) equipped with an AMT eight megapixel digital camera and AMT Image Capture Engine V602 software (Advanced Microscopy Techniques). The length of 100 random mitochondria for each condition were measured and plotted.
4
Cell Culture and PBMC Isolation Reagents
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Trypsin/EDTA and media used for cell culture (DMEM, DMEM-F12, IMDM, and RPMI 1640), as well as gradient cell separation medium used for PBMCs isolation (Lymphosep), were supplied by Biowest (Nuaillé, France). Normal-melting-point (NMP) agarose, low-melting-point (LMP) agarose, Triton X-100, 3-(4,5-dimethylthiazol-2-yl)-2,3-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), 4,6-diamidino-2-phenylindole (DAPI), penicillin–streptomycin solution stabilized, fetal bovine serum (FBS), phytohaemagglutinin (PHA), phosphate-buffered saline (PBS), and N,N-Dimethylformamide (DMF) were purchased from Sigma-Aldrich (St Louis, MO, USA). Microplates, as well as 96-well and 12-well plates, were supplied by Thermo Fisher Scientific (Waltham, MA, USA). Other chemicals used in the experiments: fluoromount-G (Invitrogen, Carlsbad, CA, USA); bleomycin (TCI); SDS (ROTH); paraformaldehyde (Polysciences, Inc; 400 Valley Rd, Warrington, PA 18976, USA); goat serum (Abcam, Cambridge, UK); the primary antibody, anti-gamma-H2AX (phospho-Ser139) (Abcam, Cambridge, UK); the secondary Goat anti-Mouse IgG Cross-adsorbed antibody, Alexa Fluor 488 p (Invitrogen, Carlsbad, CA, USA).
5
Quantification of Extracellular Vesicle Surface Markers
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Cell surface receptor expression was measured with CD69 (APC), or CD45 (PE) antibodies (BD Biosciences) using the manufacturer’s recommendations. Cells were incubated on ice for 1 hour, washed 3 times with PBS and fixed in 2% paraformaldehyde (Polysciences). Purified extracellular cellular vesicles (EV) were stained with either anti-CD63 exo-flow staining kit (Systems Biosciences) or CFSE dye (5μM) for 15 minutes at 37°C. EVs were washed in PBS four times and concentrated using Amicon 100K filter units (Millipore). Data was acquired on BD LSR II flow cytometer using single stained CompBeads (BD Biosciences) for compensation. At least 10,000 total events were collected in each experiment and the FlowJo software program (Tree Star Inc.) was used for data analysis. All flow cytometry experiments were repeated at least three times with consistent results.
6
Electron Microscopy Ultrastructural Analysis
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Electron microscopy images were obtained from the Molecular Microbiology Imaging Facility. For ultrastructural analysis, samples were fixed in 2% paraformaldehyde/2.5% glutaraldehyde (Polysciences Inc.) in 100 mmol/L cacodylate buffer (pH 7.2) for 1 h at room temperature. Samples were washed in cacodylate buffer and postfixed (1 h) in 1% osmium tetroxide (Polysciences Inc.). Samples were rinsed extensively in deionized H2O before en bloc staining (1 h) with 1% aqueous uranyl acetate (Ted Pella Inc.). After several rinses, the samples were dehydrated in a graded series of ethanol and embedded in Eponate 12 resin (Ted Pella Inc.). Sections (95 nm) (Ultracut UCT ultramicrotome; Leica Microsystems Inc.) were stained with uranyl acetate and lead citrate and viewed on a JEOL 1200 EX transmission electron microscope (JEOL USA Inc.), equipped with an 8-megapixel digital camera (Advanced Microscopy Techniques).
7
Screening TLR Pathway Genes in Cell Lines
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TLR pathway genes were targeted with 6 unique siRNA sequences (3 each from Ambion and Qiagen) per gene (Supplementary Table S1 ) and individual siRNAs were arrayed in 384-well plates. Negative control siRNAs were from Dharmacon (Non-targeting controls; NTC#2, 3 or 5). LPS contamination of the TLR ligand panel was tested using siGenome SMARTpool siRNAs against human and mouse TLR4 (GE Healthcare, Dharmacon). After siRNA transfection following the optimized protocols above, THP1 B5 cells were stimulated with TLR ligands for 4 hr and subjected to dual luciferase assay. Replicate siRNA plates were run for RAW G9 cells to permit TLR ligand treatment of one plate set for 40 min (NF-κB readout) and the second plate set for 16 hr (TNF-α reporter readout). RAW G9 cells were fixed in 4% paraformaldehyde (Polyscience, Inc) and nuclei were stained with 600 nM DAPI (Invitrogen) prior to high content imaging. The reporter responses were measured for cells transfected with each of the 6 individual siRNAs/gene, and the average values were calculated.
8
Fluorescence Analysis of β-Catenin Localization
9
Ultrastructural Analysis of Mouse Placenta
10
Aortic Lesion Quantification via Oil Red O
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The animals were euthanized and perfusion-fixed with 4% paraformaldehyde
(Polysciences, Inc. Cat #18814) via the left ventricle for 5 min. The lesions
located in the aorta and aortic roots were analyzed using Oil Red O staining. To
measure lesions in the aorta, the whole aorta, including the ascending arch,
thoracic and abdominal segments, was dissected, gently cleaned of adventitial
tissue and stained with Oil Red O (Sigma O0625)4 (link). The surface lesion area was quantified
with ImageJ software (NIH). To measure lesions in the aortic root, the heart and
proximal aorta were excised and the apex and lower half of the ventricles were
removed.
(Polysciences, Inc. Cat #18814) via the left ventricle for 5 min. The lesions
located in the aorta and aortic roots were analyzed using Oil Red O staining. To
measure lesions in the aorta, the whole aorta, including the ascending arch,
thoracic and abdominal segments, was dissected, gently cleaned of adventitial
tissue and stained with Oil Red O (Sigma O0625)4 (link). The surface lesion area was quantified
with ImageJ software (NIH). To measure lesions in the aortic root, the heart and
proximal aorta were excised and the apex and lower half of the ventricles were
removed.
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