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arrow14Double Label Immunohistochemistry For The Separate Observation Of Two Antigens Using Epipolarization Microscopy For The Immunogold-Silver Technique And Fluorescence Microscopy For The Alkaline Phosphatase Staining

C.M. van der Loos, PhD Academical Medical Center, Dept. of Cardiovascular Pathology H0-120 Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands


For immunohistochemical double staining basically two approaches exist, either doubleimmunofluorescence techniques or double-immunoenzyme detection methods. Both approaches, however, have disadvantages.

(i) The fading of the fluorescence signal at excitation and during storage. (ii) Thus far it has been proven impossible in immunoenzyme double staining methods to examine one enzymatic reaction product, singling out the other precipitate, as can be done with two fluorochromes. Despite the fact that conditions can be created to optimize the contrast between the two basic colours and the mixed-colour at sites of co-localization (Van der Loos et al., 1993), specimens cannot be examined or photographically recorded for their individual colours, not even with the aid of monochromatic filters. Most successful in this respect, but still not ideal, is the combination of an immunoenzyme method with the immunogold-silver (IGS) technique. This approach offers the possibility to observe both enzymatic reaction products and silver precipitate with bright-field microscopy, while the silver precipitate can be visualized also with epipolarization microscopy. The latter technique will not reveal the enzymatic reaction product. An obvious disadvantage is the high costs involved with full-colour reproduction.

(iii) The localization of enzymatic reaction products usually is more diffuse and less well defined compared to the staining accuracy of fluorochromes or that of a silver precipitate (Van der Loos, personal observation).

What’s New?

Alkaline phosphatase (AP) reaction products, using Fast Red TR as chromogen, show unique fluorescent properties. Minute amounts of the reaction product produce an intense red fluorescence signal with hardly any fading when excited by green light (Murdoch et al., 1990; Speel et al., 1992). Since it is well known that a silver precipitate can be studied with epipolarization microscopy, the idea was born to combine the fluorescent AP method with the IGS technique. It appeared, however, that Fast Red TR also produced a strong epipolarization image. Therefore we searched for a different chromogen. The ideal conditions were fulfilled completely by the Becton Dickinson CAS Red AP activity visualization kit (Van der Loos and Becker, 1994). Later, the Vector Red kit (Vector Laboratories) for AP activity detection, showed identical characteristics (van der Loos, personal observation). This set-up opened the possibility to perform an immunohistochemical double staining procedure, combining fluorescence (AP) with epipolarization (IGS) microscopy for the detection of the two reaction products in one and the same section.

The Protocol

Antisera, antibody/enzyme conjugates and streptavidin reagents were diluted in Tris/HCl (50 mM, pH 7.8) buffered saline (TBS) + 1% Bovine Serum Albumin (BSA). TBS washings were performed between all steps (3 x 2 min) and all incubations were performed at room temperature. Antibody/Ultra Small Gold (USG) conjugates were diluted in TBS + 0.8% BSA + 0.1% cold water fish skin gelatine. This solution was also used for washing prior to and after the incubation with the antibody/USG conjugate (3 x 10 min).

  1. Complete an appropriate double staining protocol (van der Loos et al., 1993) on either acetone fixed cryostat sections, cytospins, cell cultures, imprints etc. or routinely formalin fixed and paraffin embedded sections. The double staining protocol requires the use of an alkaline phosphatase (AP-)conjugated reagent for one antigen and an Ultra Small Gold (USG-) labeled reagent for the other antigen.
  2. Final washing with saline buffered with Tris/HCl (50 mM, pH 7.8).
  3. AP activity is detected using the CAS Red kit (Beckton Dickinson) or Vector Red kit according to the manufacturer's instructions. 1µl of 1 M Levamisole is added for inhibition of endogenous AP activity (Borgers, 1973). The total solution is cleared through a Milex-AA 0.8 µm filter and directly brought on the specimens. The formation of the AP reaction product is checked with bright-field microscopy at a low magnification. When a pink/red reaction product becomes visible, the reaction is stopped in running tap water. This reaction should be preferably completed within 5 min at room temperature, in order to keep the formation of non-specific background staining at a minimal level.
  4. Wash specimen in running tap water for 10 min and immerse in distilled water.
  5. Perform silver enhancement using the freshly prepared Aurion R-gent reagent. The reaction is checked with bright-field microscopy at low magnification. When a weak brownish precipitate becomes visible (5- 20 min), silver enhancement is stopped by immersing the specimens in distilled water.
  6. Wash with running tap water (10 min).
  7. Optional: short nuclear counterstain with haematoxilin, followed by running tap water.
  8. Immerse specimens in distilled water and mount with Glycergel (DAKO).
  9. Observe IGS reaction product with epipolarization filter and AP reaction product with standard rhodamine filter set. Reduce excitation light as much as possible with a grey filter.
  10. Photographic recording in black-and-white using Ilford HP5 (routinely processed) or in colour using Kodak EL 400 (routinely processed). For both 400 ASA film types the epipolarization and fluorescence images are individually exposed automatically at 1600-3200 ASA. For double exposure using Kodak EL 400, the images are individually exposed automatically at 3200 ASA.


This Newsflyer presents a new method for the detection of two antigens in one section. The technique is based on the combination of a fluorescent AP activity detection method and the IGS technique. The AP reaction product shows an intense red fluorescence on green light excitation, but no signal with epipolarization illumination. The IGS technique produces a distinct signal with epipolarization, but no fluorescence signal. This allows individual examination and photographic recording of two antigens, even with black-and-white photography (Van der Loos and Becker, 1994). The stability of the fluorescent AP reaction product appeared to be superior compared with traditional fluorochromes. At the time of writing this Newsflyer, our oldest specimens have been stored at room temperature for 24 months and still exhibit a bright fluorescence. Fading of the fluorescence image with high intensity excitation light is reduced using a grey filter. The stability of the IGS reaction product (a silver precipitate) during storage at room temperature or at epi-illumination with polarized UV light is beyond dispute. Specimens, which underwent single IGS staining mounted either with Glycergel (DAKO) or an organic medium, are stored for 5 years or longer without loosing their original epipolarization image (Van der Loos, personal observation).


The author is highly indebted to Professor AE Becker for his encouragement and valuable advice in preparing this manuscript.


Borgers M: The cytochemical application of new potent inhibitors of alkaline phosphatase. J Histochem Cytochem 21: 812, 1973

Murdoch A, Jenkinson EJ, Johnson GD, Owen JJT: Alkaline phosphatase-Fast Red, a new fluorescent label. Application in double labelling for cell surface antigen and cell cycle analysis. J Imm Meth 132:45, 1990 Speel JM, Schutte B, Wiegant J, Raemakers FCS, Hopman AHN: A novel fluorescence detection method for in situ hybridization, based on the alkaline phosphatase-Fast Red reaction. J Histochem Cytochem 40: 1299, 1992

Van der Loos CM, Becker AE, Van den Oord JJ: Practical suggestions for successful immunoenzyme double-staining experiments. Histochem J 25:1, 1993

Van der Loos CM and Becker AE: Double epi-illumination microscopy with separate visualization of two antigens. A combination of epi-polarization for immunogold-silver staining and epi-fluorescence for alkaline phosphatase staining. J Histochem Cytochem 42:289, 1994

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