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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% Ethanol10 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:Xylene10-15 min
    1:1 Ethanol:Xylene10-15 min
    1:2 Ethanol:Xylene10-15 min
    100% Xylene10-15 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 : Paraffin30 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).

​​

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
  • Partially fill dry ice container with dry ice and add methanol to create a cool bath, let sit.
  • Label TissueTek wells with each animal number and fill with OCT (TissueTek) freezing compound.
  • Remove excess sucrose from tissue by blotting on laboratory cleaning tissue (e.g. Kimwipes™​) and place tissue in center of well filled with OCT.
  • Orient tissue into the bottom of the well and freeze by floating on methanol bath.


    CAUTION: do not get methanol on the OCT, it will not freeze correctly.

  • Place frozen tissue blocks in -20°C freezer after they are frozen.
  • The tissue blocks are ready to be sliced after they are frozen completely.


    Do not store slides in the cryostat overnight, they will dry out and be no good. It is also a good idea to place all tissue into plastic bags in the -20°C frost free freezer to reduce drying during storage.

  • Slice sections on the cryostat.

    •  Glycol methacrylate (GMA) embedding

    Advantages of using GMA

    • ​Water miscible, doesn't require dehydration and rehydration steps.
    • No need to eliminate resin before staining. 
    • Low viscosity, penetrates tissue easily.
    • No crosslinking, no antigen retrieval.
    • Good antigen presentation.
    • Good morphology preservation (cellular localization).
    • Low temperature processing.
    • Can cut to very thin sections (1-2 µm) making the most of very small biopsies - very good resolution.


    Fixation

    Several methods of tissue fixation can be used with GMA.
    ​Fixing in acetone usually gives good results. Use the following method:

    1. Place biopsy immediately in ice cold acetone containing protease inhibitors.
    2. Fix overnight at -20°C.
    3. Replace fixative with acetone (at room temperature) 15 min.


    Processing

    1. Place biopsy in methyl benzoate for 15 min (this helps infiltration of GMA into the tissue).
    2. Place biopsy in 5% methyl benzoate in GMA 4°C.
    3. Repeat step twice more with fresh solution.


    Embedding

    Follow the kit manufacturer's instructions for embedding into GMA itself. The GMA will need to be polymerized using a catalyst (provided in commercially available kits) and left to set for 48 hr at 4°C.

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