(gentle music)
- [Narrator] Whole-cell patch-clamp recordings are made
from neurons using an intracellular recording solution
containing 50-micromolar Alexa Fluor 633.
Thus, only the recorded neuron will be filled
with a fluorescent dye that will be subsequently visualized
on the confocal microscope following the treatment
of the slice with the following protocol.
Remove the brain slice from the recording chamber
and submerge overnight in 4% paraformaldehyde
in 0.1-molar phosphate buffer pH 7.4.
Prepare 50-millimolar Tris buffer saline solution,
and add 1% Triton X-100 to a proportion of the solution.
Put a magnetic stirrer in the beaker,
and stir until the Triton has gone into solution.
Carefully remove the 4% PFA solution
without disturbing the brain slice.
Wash it with the 1% Tris-Triton solution
for 10 minutes under agitation.
Repeat this step two more times
until you have performed three washes.
Prepare the blocking solution by adding 4% normal goat serum
to 1% Tris-Triton solution.
Carefully remove the 1% Tris-Triton solution
from the last wash,
and add the blocking solution to the brain slice.
Leave to block for one hour with agitation.
Prepare the primary antibody solution
by diluting the primary antibody in the Tris-Triton solution
to the correct dilution factor.
A good starting point is the guideline provided
by Abcam with the product, but it is recommended
to optimize this for each antibody used.
Remove the blocking solution, do not wash,
and add the primary antibody solution to the brain slice.
Leave it to incubate overnight
under agitation at four degrees C.
Carefully remove the primary antibody solution.
Wash it with the 1% Tris-Triton solution
for 10 minutes with agitation.
Repeat this step two more times
until you have performed three washes.
Prepare the secondary antibody solution
by diluting the secondary antibody
in the Tris-Triton solution to the correct dilution factor.
A good starting point is the guideline provided
by Abcam with the product, but it is recommended
to optimize this for each antibody used.
Here, we are using a secondary
that is conjugated with Alexa Fluor 647.
Remove the wash solution,
and add the secondary antibody solution to the brain slice,
leaving it to incubate at room temperature for one hour.
Carefully remove the secondary antibody solution.
Wash it with the 1% Tris-Triton solution
for 10 minutes under agitation.
Repeat this step two more times
until you have performed three washes.
Carefully place the slice right side up on the slide,
and position it with a small brush.
Apply the mounting media and coverslip.
Use clear nail polish to seal the coverslip.