OpenLegislation

Division II.5 Immunohistochemistry (IHC)

United Kingdom

Original text. Later amendments are not shown.

Polyclonal antibody against ISAV nucleoprotein shall be used on paraffin sections from formalin-fixed tissue. The organs to be examined shall be mid-kidney and heart (transitional area including all three chambers and valves). Suspected cases due to pathological signs shall be verified with a positive IHC. Histological sections shall be prepared in accordance with standard methods.

  1. (1)

    Preparation of tissue sections

    The tissues shall be fixed in neutral phosphate-buffered 10 % formalin for at least 1 day, dehydrated in a graded ethanol series, cleared in xylene and embedded in paraffin, according to standard protocols. Approximately 5 μm thick sections (for IHC placed on poly-L-lysine-coated slides) shall be heated at 56 °C to 58 °C (maximum 60 °C) for 20 minutes, dewaxed in xylene, rehydrated through a graded ethanol series, and stained with haematoxylin and eosin for pathomorphology and IHC in accordance with point (2).

  2. (2)

    Staining procedure for IHC

    All incubations shall be carried out at room temperature on a rocking platform, unless otherwise provided for in this decision:

    1. (a)

      antigen retrieval shall be done by boiling sections in 0,1 M citrate buffer pH 6,0 for 2 × 6 minutes followed by blocking with 5 % non-fat dry milk and 2 % goat serum in 50 mM TBS (TBS; Tris/HCl 50 mM, NaCl 150 mM, pH 7,6) for 20 minutes;

    2. (b)

      sections shall then be incubated overnight with primary antibody (monospecific rabbit antibody against ISAV nucleoprotein) diluted in TBS with 1 % non-fat dry milk, followed by three washes in TBS with 0,1 % Tween 20;

    3. (c)

      for detection of bound antibodies, sections shall be incubated with Alkaline phosphatase-conjugated antibodies to rabbit IgG for 60 minutes. Following a final wash, Fast Red (1 mg ml– 1) and Naphthol AS-MX phosphate (0,2 mg ml– 1) with 1 mM Levamisole in 0,1 M TBS (pH 8,2) shall be added to develop for 20 minutes. Sections shall then be washed in tap water before counterstaining with Harris haematoxylin and mounted in aqueous mounting medium. ISAV positive and ISAV negative tissue sections shall be included as controls in every setup.

  3. (3)

    Interpretation of the result of IHC

    The interpretation of the result of the IHC test shall be as set out in points (a) and (b):

    1. (a)

      control sections shall be considered as positive, when it is observed that the control sections have clearly visible red-coloured (reddish) cytoplasmic and intranuclear staining of endothelia cells in blood vessels of endocardium. A test sample section shall only be regarded as positive if such clear, intranuclear red staining of endothelial cells is found;

    2. (b)

      control sections shall be considered as negative if they don't have any significant colour reaction.

    Since the intranuclear localisation is particular to the orthomyxovirus nucleoprotein during a stage of virus replication but concurrent cytoplasmic staining is often dominant, cytoplasmic and other staining patterns without intranuclear localisation shall be considered as nonspecific or inconclusive.

    The strongest positive staining reactions are usually obtained in endothelial cells of heart and kidney. Endothelial staining reactions within very extensive haemorrhagic lesions may be slight or absent, possibly because of lysis of infected endothelial cells.