- The water provides a humid environment for the coverslips
when they are being incubated with primary/secondary anti-
body, thus ensuring they do not dry out. - We used rabbit anti-NFκB (p65) (#8242 from Cell Signaling
Technologies). If assessing phospho-JNK localization, use
mouse anti-phospho-JNK1/2 (#9255 from Cell Signaling
Technologies works well) diluted 1:400 in IF buffer. - When pipetting antibody ensure that enough space is left
between replicates/samples so that they do not end up merg-
ing together. - Reverse pipetting consists of pushing the pipette plunger down
to the second stop, drawing up desired volume, and dispensing
liquid by depressing pipette plunger down to the first stop. The
purpose of this is to reduce the risk of bubbles which would
affect exposure of coverslip to antibody. - Incubating coverslips in this way dramatically reduces the vol-
ume of antibody required, compared with performing incuba-
tions in the well. In the event that there is not even coverage of
the coverslip surface, pick the coverslip up and gently wipe
away the liquid from the parafilm using tissue. Pipette another
droplet of antibody and try again. It is important that you
pipette the antibody onto dry parafilm; otherwise it is less likely
to sit as a neat drop. - Covering in this way helps maintain a humid environment for
the coverslips. Cover a box or tub in tinfoil so it can function to
protect coverslips from light while incubating in fluorescent
secondary antibody. - We used Alexa Fluor®488-linked goat anti-rabbit antibodies.
If using mouse anti-phospho-JNK1/2 antibodies as the pri-
mary antibody, use Alexa Fluor®conjugated goat anti-mouse
IgG as a secondary antibody (Invitrogen, Life Technologies
Ltd). - Try to minimize the presence of bubbles when spotting
mounting agent. Only spot the mounting agent on one or
two microscope slides at a time to prevent it drying out before
the coverslip is placed over it. Two or three spots of mounting
agent and therefore two or three coverslips can be mounted on
each slide. - The protocol describes collection of simple conditioned media
which can be used for ELISA or multiple cytokine analysis, as
we have described previously [12]. If that conditioned media
are intended to be collected and used to examine the influence
of secreted cytokines/chemokines on the behavior of other
cells, it is important to ensure that AMPK activators and
pro-inflammatory stimuli are washed away such that those
Roles in Inflammation 317