2008 PBL Cat_mech_r.qxd:PBL Catalog.mech 11/29/07 1:51 PM Page 47
Technical Appendix
Direct versus Indirect Immunofluorescence Cell Surface Staining
Cell surface markers are mainly proteins associated to the extracellular
Direct immunofluorescence of fixed cells
part of the cell membrane. PBL InterferonSource offers a variety of
Cell Preparation
reagents to characterize cytokine cell surface markers.
Before selecting which procedure to use consider the following:
1. Seed 1 x 10
5
cells per cover slip in a 24 well-plate and grow
sensitivity, time and the cost of each method including the level of
cells to 70-90% confluency.
experience in conjugating a fluorochrome to antibody.
2. Wash cells once with 1x PBS.
Direct Method
The simplest technique is the direct method, which uses a fluorophore
3. Cover cell layer with 200-500 µl of ice-cold acetone. Incubate 5
o
conjugated primary antibody that recognizes and binds to the cell surface
minutes at -20 C.
antigen. The direct method requires no labeling of antibody, less
incubation time and results can be obtained within 30 minutes using flow
4. Aspirate solution and wash cells once with 1x PBS.
cytometry or immunohistochemistry. They are also advantageous when
performing multicolour immunofluorescence analysis or using several
5. Block cell layer with 5% normal goat serum containing 1% Triton X-100
antibodies of the same isotype.
for 30 minutes at room temperature.
Indirect Method
Stain Cells With Antibody
In the indirect method, cells are incubated with an unlabeled primary
antibody. The primary antibody binds to cell surface antigens. For
1. Dilute monoclonal FITC-conjugate antibody 1:10 in 1x PBS
detection a fluorophore-conjugated secondary antibody which attaches to
the primary antibody is added.
2. Add diluted antibody sufficient to cover the cell layer and incubate
cover slip for 1 hour at room temperature-away from direct light.
Detection
After staining the cell sample is analyzed by flow cytometry to enumerate
3. Aspirate the solution and add 5 µg/ml Hoechst 33342 (Invitrogen)
cells expressing the antigen, and estimate the quantity of antigens per cell
for 10 minutes at room temperature.
(intensity of fluorescence).
4. Rinse cover slip 3 times with PBS, 3 minutes between
Controls
each rinse.
Controls are used to determine the specificity of the antibody used for the
assay. Both negative and positive control samples should be included in
5. Invert cover slip onto 15 µl mounting medium, previously
any given study when possible.
applied to slides, using forceps.
Antibody Dilution and Incubation Time
6. Analyze the cover slip using fluorescence microscopy.
The optimal antibody dilution and incubation time is empirically determined
Store cover slips in the dark at 2 to 8
o
C.
for any given set of experimental conditions. The conditions chosen are
For longer storage, apply nail polish to the edges of the
those that demonstrate the maximum specific signal and having the lowest
cover slip.
background. To assess the working antibody dilution and incubation times to
use, select one parameter to vary per study. For example, chose a fixed
NOTE: These applications are provided as a general guide.
incubation time and prepare a titration series of antibody to test.
Levels of IFN receptors and IFN proteins can vary depending
on cell type and experimental treatments. Additional studies
Positive Controls
may be necessary to ensure optimal results.
A positive control sample may be any tissue, cell line, or a purified
molecule, that is known to contain the antigen of interest.
Negative Controls
Negative controls are used to evaluate nonspecific binding or to
discriminate specific staining from artifacts Perform an experiment
excluding the primary antibody. Incubate samples without the primary
antibody. Use normal serum or an isotype control (for monoclonal
antibody) from the same species as primary antibody. Alternatively, the
use of unstained cells can be used. For intracellular cytokine staining, the
use of blocking controls is recommended.
If purified antigen can be obtained, perform an inhibition study. Addition
of the purified antigen along with the primary antibody should inhibit
signal but the addition of unrelated antigens should not.
www.interferonsource.com Tel: +1 732-777-9123
I
Fax: +1 732-777-9141
47
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