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.

Contents

​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 three times, 20 mins per exchange.


    4. Exchange xylene with paraffin twice, for 1 - 2 hrs per exchange.


    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. Incubate sample with sucrose solutions in the following sequence.

      SolutionIncubation time
      10% Sucrose15 min or until sample drops to the bottom of the vial
      30% Sucrose15 min or until sample drops to the bottom of the vial
    3. Partially fill dry ice container with dry ice and add methanol to create a cool bath, let sit.
    4. Label Tissue Tek wells with each animal number and fill with OTC (TissueTek)
      freezing compound.
      Label Tissue Tek wells with each animal number and fill with OTC (TissueTek)freezing compound. 
    5. Remove excess sucrose from the tissue by blotting on Kimwipes and place the tissue in the center of well filled with OTC.
    6. Orient tissue into the bottom of the well and freeze by floating on methanol bath. CAUTION: do not get methanol on the OTC, it will not freeze correctly.
    7. Place frozen tissue blocks in -20°C freezer after they are frozen. 
    8. 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 frost-free freezer to reduce drying out during storage. 
    9. 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 for 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 -20°C.
    3. Replace fixative with acetone (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 times for 2 hr. 

    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 sit for 48 hr at 4°C.