Welcome to the Novus visual protocol series
In this video you will learn how to perform all phases of florescent
immunocytochemistry
using the most common methods for this assay.
Before starting the ICC procedure, we will demonstrate how to prepare
coverslips and cell culture plates followed by the plating of our cells.
We begin by acid treating new coverslips in one molar of hydrochloride acid for
24 hours.
After carefully decanting off the acid and removing the glass coverslips,
we wash each with distilled water,
then a final rinse with ethanol.
As an optional step you can place the coverslips into a subbing rack
submerge it
a .1 mg/solution gelatin or polylysine
for five minutes.
These coatings aide in providing additional adherence of cells to the
coverslips
ensuring that they are not detached during later wash steps.
After treatment remove the subbing rack and dry the coverslips in the culture
hood or a heated oven.
Clean dry coverslips are then inserted into each well of a six well
culture plate
and covered with a lid.
To save time, large volumes of plates can be prepared ahead of time for later use.
The plates can also be sterilized by placing them under the hood's UV light.
With clean coverslips prepared in six well plates,
we can now add our cells.
Platted here in cells add a density of one half million cells per well.
Cells are then cultured overnight in the incubator or until they reach your preferred
density.
With the cells plated on the previous day and cultured overnight, we are ready
to start with the ICC procedure.
On this first day of ICC, we will fix,
permeabilize,
blot and add a primary antibody to the cells.
Start by aspirating culture medium from each well followed by fixation of the
cells with 4% formaldehyde or 10% formalin
for 10 minutes at room temperature.
Aspirate fixative and rinse each well twice with ice cold PBS.
Be careful not to let the cells dry out in this
or any further step of the protocol.
If the epitope of your protein of interest is expressed intracellularly,
cellular permeabilization is necessary for the antibody to gain access to the
cell.
Although there are many different
permeabilization agents, the most common are .1-.5%
concentration
of Tween-20
or Triton-X100
diluted in PBS.
Tween-20 is used for epitopes located in cytoplasm
while Triton-X100 is used for permeabilizing the nucleus and mitochondria.
Incubate wells with permeabilization buffer for 10 minutes at room temperature.
Aspirate permeabilization buffer and wash three times for 5 minutes each with
PBS twine.
PBS twine contains .1% Tween-20.
We will now block unspecific binding sites with blocking buffer for 1 hour
at room temperature.
The best blocking buffer is PBST containing 10% serum from the host
species of your secondary antibody.
However,
1% BSA in PBST may also be used.
Prepare the primary antibody by diluting it in blocking buffer
at the recommended dilution specified by the datasheet.
Multiple tighter dilutions are beneficial to account for variables that may be
different in your assay
and for achieving the best results.
Incubate at 4 degrees Celsius overnight.
In our example,
since we are using an unconjugated primary we will be using a fluorophore
conjugated secondary on day two.
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 cell
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
with the recommend dilution specified on the datasheet
and incubated 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 second 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 further 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 will prevent photo bleaching.