- Spot 35μl of rabbit anti-NFκB (p65) antibodies diluted 1:50
in IF buffer for each coverslip onto parafilm (seeNotes 14and
15 ). Do this using a reverse pipetting technique (seeNote 16).
Leave the secondary antibody only control coverslips in the
plate. - Using hooked forceps and needle, remove coverslip from well,
dab edge gently on tissue to remove excess liquid, and lay it
over the drop of primary antibody (cell-side down) so that the
drop is sandwiched between the cells on the coverslip and the
parafilm (seeNote 17). - Repeat for all coverslips and cover them all with a box/tub (see
Note 18) and incubate for 1 h. - Using hooked forceps and needle, move each coverslip back
into the 12-well plate so that the cells are facing upward. - Wash four times with 400μl/well of IF buffer.
- Spot 35μl of Alexa Fluor®488-conjugated goat anti-rabbit
IgG antibodies and RedDot (both diluted 1:200 (seeNote 19)
in IF buffer) per coverslip onto parafilm as described in
step 17. This should now include the secondary antibody
only control coverslips. - Transfer coverslips to secondary antibody droplets as described
insteps 18and 19. - Incubate for 30 min in the dark under a box/tub.
- Using hooked forceps and needle, move each coverslip back
into the 12-well plate so that the cells are facing upward. - Repeat washstep 21.
- Add a drop of Immunomount for each coverslip onto a micro-
scope slide (seeNote 20). Using hooked forceps and needle,
remove coverslip from plate and dip in PBS. Dab the edge of
coverslip and the side lacking cells on a tissue to absorb excess
PBS and gently lay each coverslip cell-side down on a drop of
Immunomount. - Keep coverslips on slides flat, dark and undisturbed overnight,
before transferring to a slide box. - Visualize with a 63oil objective and the relevant filter by
fluorescence confocal microscopy. - Nuclear fluorescence in the resultant images can be quantified
using ImageJ software to obtain values corresponding to the
density of multiple points within the nucleus. These data can be
used to assess the mean density of nuclear fluorescence per cell.
A minimum of 50 cells should be analyzed per treatment, and
the mean fluorescence taken.
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