IMMUNOFLOURESCENCE PROCEDURES

1. Cut 46 micron thick tissue sections, and apply to slides.

2. Deparaffinize in xylenes using three changes for 5 minutes each.
3. Hydrate sections gradually through graded alcohols:
a. wash in 100% ethanol twice for 10 minutes each,
b. then 95% ethanol twice for 10 minutes each.
c. Wash in deionized H2O for 1 minute with stirring.
d. Aspirate excess liquid from slides.

4. Antigen unmasking-citrate buffer

a. Heat treatment (recommended method): Place slides in a container and cover with
10 mM sodium citrate buffer, pH 6.0
b. Heat at 95 C for 5 minutes. Top off with fresh buffer and heat at 95 C for 5
minutes (optimal incubation time may vary for each tissue type).
c. Allow slides to cool in the buffer for approximately 20 minutes.
d. Wash in deionized H2O three times for 2 minutes each. Aspirate excess liquid
from slides
5. Incubate specimens with 10% normal blocking serum (Normal Sera for
Immunohistochemistry) in PBS (Buffers and General Solutions) for 20 minutes to
suppress non-specific binding of IgG. Blocking serum ideally should be derived from the
same species in which the secondary antibodyis raised. Wash with PBS.
6. Incubate with primary antibody for 60 minutes. Optimal antibody concentration should
be determined by titration; recommended range is 0.55.0 g/ml in PBS with 1.5%
normal blocking serum. Wash with three changes of PBS for 5 minutes each.
7. Incubate for 45 minutes with either biotin-conjugated or fluorochrome-conjugated
secondary antibody (Secondary Antibodies for Immunohistochemistry) diluted to 15
g/ml in PBS with 1.5%3% normal blocking serum. Optimal antibody concentration
should be determined by titration. Wash with three changes of PBS. If fluorochrome-
conjugated secondary antibody is used, incubate in a dark chamber and omit the next
step.
8. Incubate with streptavidin-fluorescein for 15 minutes in a dark chamber. Optimal
streptavidin conjugate concentration for a given application should be determined by
titration; recommended range is 1020 g/ml in PBS. Wash extensively with PBS.
9. Mount coverslip with UltraCruz Mounting Medium