Biology Reference
In-Depth Information
FIGURE 1.3
Overview of SpIDA procedures.
the linear regime of the PMT detectors (e.g., avoid saturation and nonlinear
PMT response) of the desired system. To date, SpIDA has been successfully applied
to quantify mCherry- and GFP-tagged proteins along with proteins visualized by
immunofluorescence staining with Alexa Fluor 488, 546, 633, and 647 antibody
conjugates. For fluorophore-conjugated antibodies, it is important to use versions
that have a high number of fluorescent moieties/antibody molecules. This informa-
tion is available from data sheet specific for the antibody lot used. Indeed, we have
observed that a higher degree of labeling per antibody yields less variation in the
establishment of the monomeric QB. This distribution can also be taken into account
during the analysis if needed (
Godin et al., 2011
).
1.3.1.1
Sample preparation
As cell fixation and immunochemistry are techniques routinely used in most cell bi-
ology laboratories and are the subject of many protocol papers and manuals, we will
not go into details for these steps (
Maity, Sheff, & Fisher, 2013
). We will focus on
image acquisition and SpIDA analysis. Just remember to choose fluorophores ac-
cordingly to system performance.
In the case of an upright microscope, cells are grown on glass coverslips and
mounted with antifading reagent following fixation. For an inverted microscope,
cells may be grown on glass bottom plates (e.g., MatTek culture dishes), chemically
fixed (e.g., paraformaldehyde, glutaraldehyde, or methanol) and immersed with
