IHC for brain slice sections video protocol

Learn how to visualize your proteins of interest with IHC on brain slice sections after electrophysiology recording (4:35 minutes).

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In this protocol, whole cell patch-clamp recordings were made from neurons using an intracellular recording solution containing 50 µM Alexa Fluor® 633. Thus, only the recorded neurone will be filled with a fluorescent dye that will subsequently be visualized on the confocal microscope following the treatment of the slice with this protocol.

Tissue fixation

Remove the brain slice from the recording chamber and submerge overnight in 4% PFA (paraformaldehyde) in 0.1 M phosphate buffer pH 7.4. 

Day 1

Preparation of solutions

  1. Prepare 50 mM TRIS buffered saline and add 1 % Triton X-100 to a proportion of the solution.
  2. Stir with magnetic stirrer until triton has gone into solution.


Rinsing of tissues in solution

  1. Carefully remove the 4% PFA solution without disturbing the brain slice.
  2. Wash it with the 1% Tris-Triton solution for 10 minutes under agitation.
  3. Repeat this step 2 more times until you have performed 3 washes.


Blocking with NGS for 1 hour with agitation

  1. Prepare blocking solution by adding 4% normal goat serum to 1% Tris-Triton solution.
  2. Carefully remove the 1% Tris-Triton solution from the last wash and add the blocking solution to the brain slice.
  3. Leave to block for 1 hour with agitation.


Addition of primary antibody

  1. 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 with the product but it is recommended to optimize this for each antibody used).
  2. Remove the blocking solution and add the primary antibody solution to the brain slice.
  3. Incubate overnight under agitation at 4oC.


Day 2

Rinsing of tissues in solution

  1. Carefully remove the 4% PFA solution without disturbing the brain slice.
  2. Wash it with the 1% Tris-Triton solution for 10 minutes under agitation.
  3. Repeat this step 2 more times until you have performed 3 washes.


Addition of secondary antibody

  1. 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 with the product but it is recommended to optimize this for each antibody used).
  2. For this protocol we used a secondary antibody that is conjugated with Alexa Fluor® 647.
  3. Remove the wash solution and add the secondary antibody solution to the brain slice, leaving it to incubate at room temperature for 1 hour.


Cover slipping

  1. Carefully remove the secondary antibody solution.
  2. Wash it with the TRIS buffered saline solution for 10 minutes with agitation.
  3. Repeat this step 2 more times until you have performed 3 washes.
  4. Carefully place the slice (right side up) on the slide and position it with a small brush.
  5. Apply mounting media and coverslip.


Once coverslip has been applied, the sample is ready for visualization with the confocal microscope.


Acknowledgment

The video protocol was produced by Abcam in partnership with NeuroSolutions.

Alexa Fluor® is a registered trademark of Life Technologies. Alexa Fluor® dye conjugates contain(s) technology licensed to Abcam by Life Technologies.




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