Fab blocking of endogenous immunoglobulins on mouse tissue – example protocol

Preparation of sample material: paraffin sections: rehydration and antigen demasking according to recommendations of primary antibody manufacturer, frozen sections: fixation and washing in buffer. Cells: washing, fixation and washing.

  1. Wash in TBS for 2 x 5 minutes.
  2. Optional: cleaning of tissue sections by incubation with 1% TX-100 in TBS for 30 minutes at room temperature.
  3. Removal of endogenous peroxidase activity in 0,6% H2O2 in TBS for 30 minutes at room temperature.
  4. Note: alternatively apply ready to use peroxidase blocking solution from dianova (ACA500(IVD), ACA999(IVD)).
  5. Wash in TBS for 3 x 5 minutes.
  6. Blocking with normal serum: incubate cells / tissue with 5% normal serum in TBS (+ 0,3% TX-100) for 30 minutes at room temperature, in order to block unspecific binding sides for preceeding antibodies in the tissue sample.
    Note: normal serum should be the same species than the host species of the secondary antibody (e. g. goat normal serum, cat.# 005-000-121).
  7. Wash in TBS for 3 x 5 minutes.
  8. Blocking of endogenous mouse immunoglobulins: incubate cells / tissues with 20µg – 100 µg/ml unconjugated Fab fragments anti-mouse IgG (H+L) in TBS for 1 hour at room temperature.
    Note: 1. Fab fragments should be the same species than host species of the secondary antibody (e. g. Fab goat anti-mouse IgG (H+L), cat.# 115-007-003). 2. The blocking efficiency can be increased over the concentration of the Fab fragments and over the incubation time (2 hours at room temperature or over night at 4°C).
  9. Wash in TBS for 3 x 5 minutes.
  10. Primary antibody incubation: incubate cells / tissue with mouse primary antibody diluted according to manufacturer’s recommendation for 30 – 60 minutes at room temperature. Dilution in TBS/1%BSA or in normal serum blocking solution (5.).
  11. Wash in TBS for 3 x 5 minutes.
  12. Secondary antibody incubation: incubate cells / tissue with peroxidase conjugated secondary antibody anti-mouse IgG (H+L) in TBS according to the recommendation of the manufacturer (approx. 0,2 – 2 µg/ml) for 30 – 60 minutes at room temperature (e. g. goat anti-mouse IgG (H+L),cat.# 115-035-062, 115-035-146, 115-035-166).
  13. Wash in TBS for 3 x 5 minutes.
  14. Incubation with Substrate: for 3 – 7 minutes in chromogen substrate solution (e. g. DAB) at room temperature (cat.ACH500 (IVD), ACT500 (High Contrast, IVD)).
  15. Rinse in tap water.
  16. Counterstain with hematoxylin as desired (cat.# HAQ500).
  17. Rinse in distilled water for 3 x 5 minutes.
  18. Let air dry and coverslip with permanent mounting medium (cat.#  PMT030)
Notes:
  1. Instead of TBS as buffer for blocking, wash and antibody dilution solution also PBS can be used. However, for a reduced background TBS may be more favourable than PBS.
  2. Background staining cannot only be due to a single cause. Among others, also activity of endogenous peroxidases in the sample tissue or Fc receptors may have an influence. Peroxidase activity is inhibited by a blocking step before the incubation with normal serum or after the primary antibody incubation. The binding of the secondary antibody to Fc receptors on cells and tissues can often be inhibited by a suitable blocking with normal serum or with special Fc receptor blocking solutions but also it can most effeciently be prevented by the use of F(ab’)2 fragment secondary antibodies (e. g. in step 11. F(ab’)2 Goat anti-Mouse IgG(H+L), cat.# 115-036-062).