IHC tissue processing protocol

Once the tissue is fixed, it needs to be processed so that it is adequately supported for cutting into sections of up to 5 µm thickness.

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The tissue is dehydrated, cleared and then infiltrated with medium to enable sectioning. Paraffin wax is the most common medium used for immunostaining.

Paraffin tissue processing

  1. After fixation, rinse tissue with PBS until fixative is completely removed. 
  2. Dehydrate tissue using ethanol in the following sequence:

    SolutionIncubation time
    50% Ethanol
    		10 min
    70% Ethanol10 min
    80% Ethanol10 min
    95% Ethanol10 min
    100% Ethanol10 min
    100% Ethanol10 min
    100% Ethanol10 min
  3. Exchange ethanol with xylene in the following sequence:

    SolutionIncubation time
    2:1 Ethanol:Xylene
    		10-15 min
    1:1 Ethanol:Xylene10-15 min
    1:2 Ethanol:Xylene10-15 min
    100% Xylene
    		10-15 min
    100% Xylene10-15 min
    100% Xylene10-15 min
  4. Exchange xylene with paraffin. The following steps are done in a vacuum oven set for 54-58°C. Paraplast X-tra™ or Paraplast Plus™ (the latter has DMSO added to facilitate infiltration) can be used. Do not let the paraffin exceed 60°C for prolong periods of time because this will degrade the paraffin polymers and make it hard and brittle.

    SolutionIncubation time
    2:1 Xylene : Paraffin
    		30 min
    1:1 Xylene : Paraffin30 min
    1:2 Xylene : Paraffin30 min 
    100% Paraffin1-2 hr
    100% Paraffin1-2 hr or overnight
  5. Embed in fresh new paraffin and orient tissue as desired before it hardens (vertical for embryos).

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Frozen tissue embedding

  1. Tissue ready for processing should be fixed and stored in PBS.
  2. Sucrose infiltration.

    SolutionIncubation time
    10% Sucrose15 min or until sample drops to bottom of vial
    2:1 10% Sucrose : 30% Sucrose

    15 min or until sample drops to bottom of vial

    1:1 10% Sucrose : 30% Sucrose15 min or until sample drops to bottom of vial
    1:2 10% Sucrose : 30% Sucrose15 min or until sample drops to bottom of vial
    30% Sucrose15 min or until sample drops to bottom of vial
    30% Sucrose15 min or until sample drops to bottom of vial
    30% Sucrose15 min or until sample drops to bottom of vial
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