Five-week-old female BALB/cCrSlc mice (SLC, Hamamatsu, Japan) were injected with 105 focus forming units (FFU) of KHF4 or KHF5 via the tail vein. The outline of animal experimentation is shown in Supplementary Figure S1 . The weights of the mice were measured from 1 to 14 dpi. At 1, 3, 5, and 7 dpi, two animals were euthanized and blood was collected. If available, urine samples were collected. The lungs, kidneys, spleens, and livers were collected and stored at −80 °C for RNA extraction and viral protein detection. Tissue pieces from the lungs were used for further cultivation to analyze viral replication. Mouse organs were fixed in 4% phosphate-buffered paraformaldehyde (Fujifilm Wako) for histological analysis. All animal experiments were approved by the Animal Studies Ethics Committee of Hokkaido University (19-0088). The mice were treated according to the laboratory animal control guidelines of the Hokkaido University Institutional Animal Care and Use Committee. Experiments involving viral infections were performed at a BSL-3 facility.
Phosphate buffered paraformaldehyde
Manufactured by Fujifilm
Sourced in Japan
Phosphate-buffered paraformaldehyde is a chemical compound used in laboratory settings. It serves as a fixative, preserving the structural integrity of biological samples for further analysis and processing.
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Lab products found in correlation
3 protocols using phosphate buffered paraformaldehyde
1
Mouse Model of Viral Infection
2
Histological Analysis of Murine Lung Tissue
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Lungs were harvested to prepare paraffin-embedded sections for histological analysis. PBS was perfused through the lungs of mice under deep anesthesia, and the lungs were then fixed by intratracheal instillation of phosphate-buffered paraformaldehyde (Wako, Osaka, Japan). Airway EC areas were evaluated in five randomly selected sections, and each section was subjected to hematoxylin and eosin staining and immunostaining, as previously described [14 (link), 15 (link)].
3
Alexa 555-CRP Binding Assay with CL-P1 and LOX-1
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The binding of Alexa 555-CRP with full length CL-P1, CL-P1 deletion mutants and LOX-1 was performed as previously described (10) . In brief, 24 h after transfection cells were washed twice with ice-cold HAM's F12 medium with 5% FBS. Then, the medium was replaced with ice-cold Ham's F-12/10 mM HEPES containing 10 µg/ml of Alexa 555-CRP and cells were incubated at 4 °C for 1 h.
After being washed with ice cold PBS, the cells were fixed with 4% phosphate-buffered paraformaldehyde (Wako Pure Chemical Industries). For the inhibition assay, 10 µg/ml poly(I) or poly(C) were pre-incubated with cells before the addition of Alexa 555-CRP. For the phosphocholine inhibition assay, 1 mM phosphocholine was mixed with Alexa 555-CRP and then incubated with cells for 1 h at 4 °C. The expression of CL-P1 and LOX-1 was visualized using anti-myc antibody followed by Alexa 488 anti-mouse IgG. Nuclear counterstaining was performed with Hoechst 33342 obtained from Invitrogen. The images were taken using a fluorescent microscope (BZ-9000, Keyence). Signal intensity was calculated by using the BZ-HIC program (Keyence).
After being washed with ice cold PBS, the cells were fixed with 4% phosphate-buffered paraformaldehyde (Wako Pure Chemical Industries). For the inhibition assay, 10 µg/ml poly(I) or poly(C) were pre-incubated with cells before the addition of Alexa 555-CRP. For the phosphocholine inhibition assay, 1 mM phosphocholine was mixed with Alexa 555-CRP and then incubated with cells for 1 h at 4 °C. The expression of CL-P1 and LOX-1 was visualized using anti-myc antibody followed by Alexa 488 anti-mouse IgG. Nuclear counterstaining was performed with Hoechst 33342 obtained from Invitrogen. The images were taken using a fluorescent microscope (BZ-9000, Keyence). Signal intensity was calculated by using the BZ-HIC program (Keyence).
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