All tags IHC protocols IHC permeabilization protocol

IHC permeabilization protocol

Permeabilization is only required when the antibody needs access to the inside of the cells to detect the protein. Cytoskeletal, viral and some enzyme antigens usually give optimal results when fixed with acetone, ethanol or formaldehyde (high concentration). 

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Antigens in cytoplasmic organelles and granules will require a fixation and permeabilization method depending on the antigen. Please refer to the antibody datasheet for recommendations.

Solvents

  1. Acetone fixation will also permeabilize.
  2. Methanol fixation can be used to permeabilize but is not always suitable.

    These reagents can be used to fix and permeabilize, or can be used after fixation with a crosslinking agent such as paraformaldehyde.

Detergents

  1. Triton or NP-40
    Use 0.1 to 0.2% in PBS for 10 min only.

    These will also partially dissolve the nuclear membrane and are therefore very suitable for nuclear antigen staining.



    As there are harsh detergents, they will disrupt proteins when used at higher concentrations or for longer amounts of time, affecting staining results.


  2. Tween 20, Saponin, Digitonin and Leucoperm
    Use 0.2 to 0.5% for 10-30 min.

    These are much milder membrane solubilizers. They will give large enough pores for antibodies to go through without dissolving the plasma membrane. They are suitable for antigens in the cytoplasm or the cytoplasmic face of the plasma membrane and for soluble nuclear antigens.


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