Author: Jane Mckeating
NS5A staining – indirect IF
1. Carefully remove tissue culture media from vessel
without disturbing monolayer and fix cells with prechilled methanol (stored at –20C) for 5 mins at room
temperature. For a 24-well plate, 1ml per well – for
HCVcc infections ensure that the volume of methanol
used exceeds the culture volume of the infection.
Remove methanol and store fixed cells in PBS – at this
stage the cells can be kept at 4C before staining for
several days to 1 week.
2. Block cells in PBS/1%BSA/0.1% saponin (Wash buffer-WB)
for 30 mins at room temperature.
3. Add primary antibody 9E10 mouse anti-NS5A diluted
1:200 in WB for 30 mins at Room temperature. For 24well plates, use 150ul of Ab/well.
4. Wash cells 2x PBS – taking care not to disturb the
monolayer.
5. Detect cell-bound 9E10 with anti-mouse Ig-Alexa488
diluted 1:2000 in WB. Incubate cells for 30 mins at
Room temperature.
6. Wash cells as above for step 6.
7. Observe and count foci by UV microscopy. Note cells
can be kept at 4C wrapped in foil for 1 week.