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NF-κB translocation immunofluorescence assay

RM Raymond Tshepiso Makola
JK Joe Kgaladi
GM Garland Kgosi More
PV Petrus Jansen van Vuren
JP Janusz Tadeusz Paweska
TM Thabe Moses Matsebatlela
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Raw 264.7 macrophage cells were cultured in 6 well plates on the slides at 4 × 105 cells/well for 3 h. This was followed by RVFV inoculation at 1 × 104.8 titre/mL for 1 h, and the excess virus was discarded. Cell treatment with lithium was executed as outlined in cell treatment and some wells were stimulated with LPS (5 mg/mL) for 24 h. The media was then aspirated after 24 h incubation and cells were fixated with 4% paraformaldehyde for 1 h. Thereafter, cells were permeabilised with 0.1% Triton X-100, 1%BSA for 60 min and the nonspecific binding sites were blocked by adding 1% BSA for 1 h, followed by 2× wash with wash buffer. Cells were incubated for 60 min with rabbit anti-p65 antibody (1:500) (Santa Cruiz, USA) followed by 3× wash with wash buffer. The cells were incubated with FITC-labelled goat anti-rabbit secondary anti-body for 60 min. After 5 min, the nuclear staining was done with DAPI, and then cells were mounted on slides using 50% glycerol and analysed using the fluorescent inverted Nikon Ti-E microscope at 20× magnification.

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