Tip:
Highlight text to annotate it
X
In day two of our ICC procedure we will add our secondary antibody, perform
an optional double labeling and nuclear labeling step,
mount our coverslips to slides, and obtain images of our antibodies with
a fluorescent microscope.
First
we aspirate off primary antibodies solution following by washing the cells
three times with PBST for five minutes each.
Next prepare the floraform conjugated secondary antibody which will bind to
the primary antibody.
Dilute the secondary blocking buffer
at the recommend dilution specified on the datasheet
and incubate at room temperature for one hour.
Covering the plates with foil prevents sensitive dyes from degrading.
Aspirate off the secondary antibody solution followed by washing of the cells
three times with PBST
for five minutes each.
As an optional step a separate primary and secondary pair can be used
to visualize a second epitope by using a different floor for it.
In addition, DNA binding dyes such as DAPI can be applied without the need for
secondary antibodies.
Refer to the full written Novus Protocol for instructions on how to double and
triple label.
Coverslips are now ready to be mounted onto microscope slides.
Take a clean slide and dispense one drop of antifade mounting medium by slowly
dialing down the plunger of the pipette.
Carefully remove a coverslip from the well,
allow excess wash to drip off
and place the cells face down onto the slide.
Clear fingernail polish can be used to seal the coverslip and prevent it from
drying out.
Slides can now be visualized under a microscope or stored at -20
or 4 degrees Celsius in a dark slide box or slide book.
Limiting the amount of time each slide is exposed to the microscope's light will aide
in prolonging the florescent signal
and prevent photo bleaching.