150
- Incubate the sections with caspase antibody at appropriate
dilution (at least 1:200) in antibody dilution buffer in a humid-
ifi ed chamber for 1 h at room temperature or overnight at
4 °C. To assess nonspecifi c staining or verify the binding speci-
fi cities of primary antibodies, incubate the sections in antibody
dilution buffer or equal amounts of nonspecifi c mouse, rabbit,
or goat immunoglobulins without the primary antibody for
negative controls. - Wash the sections with PBS-T for 5 min, repeat two times.
- Incubate the sections with HRP-conjugated secondary anti-
body at appropriate dilution in antibody dilution buffer at
room temperature in the dark for 1 h. - Wash the sections with PBS-T for 5 min, repeat two times.
- Incubate the sections in dark with HRP substrate solution.
- Wash slides with PBS-T for 5 min, repeat two times to remove
excess chromogen. - Dehydrate the slides with ascending graded alcohols (for 20 s
each in 35, 70, and 95 % ethanol, and 2 min in 100 % ethanol)
and clear in xylene. - Mount the slides with mounting medium.
- Incubate the sections in blocking buffer at room temperature
for 30 min ( see Note 10 ). - Incubate the sections with primary antibody at appropriate
dilution (at least 1:200) in antibody dilution buffer in a humid-
ifi ed chamber for 1 h at room temperature or overnight at
4 °C. - Wash the sections with PBS-T for 5 min, repeat two times.
- Incubate the sections with fl uorescent secondary antibody at
appropriate dilution in antibody dilution buffer at room tem-
perature in the dark for 1 h ( see Note 11 ). - Wash the sections with PBS-T for 5 min, repeat two times.
- Counterstain nuclei with DAPI in mounting medium (e.g.,
VECTASHIELD). - Coverslip and cure slides in the dark at room temperature for
4 h or at 4 °C overnight. - Record immunofl uorescence using appropriate fi lter for sec-
ondary antibody and for DAPI and record bright-fi eld or DIC
images. - Compare with sections developed without primary antibody to
assess background fl uorescence of tissue.
3.3.3.2 Immunofl uores-
cence Staining
3.3.4 Imaging
of Immunofl uorescence
( See Note 12 )
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