Biology Reference
In-Depth Information
7. Defi ne the histogram density of the spots on the fi lm for a par-
ticular cytokine in the experimental samples and calculate the
amount of the cytokine present in the sample by using calibra-
tion curves from
step 5
.
4
Notes
1. All experiments involving live animals must be reviewed and
approved by the institution animal care and use committee
prior to conducting any such experiments, including appropri-
ate methods for euthanasia.
2. This should be done independently for each different tissue of
interest, as in each one of them tissue-specifi c inhibitors of
protein-capture/developing antibody interaction can be pres-
ent at various and unpredictable levels.
3. We have done so not only to quantify those proteins of interest
which we could detect in our samples, but also for proteins that
were not evidently detected in our experimental samples to
prove that when a protein has not being detected (specifi cally
TNF-
), it was truly because the cytokine/chemokine had not
being produced, as evidenced by the fact that we could defi -
nitely establish a specifi c and sensitive standard curve for every
protein tested so far.
α
Acknowledgments
This work was supported by US National Institutes of Health
grants AI065429 and CA141439.
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