J Clin Pathol: first published as 10.1136/jcp.32.10.971 on 1 October 1979. Downloaded from

Journal of Clinical Pathology, 1979, 32, 971-978

Immunoperoxidase technique in histopathology: applications, methods, and controls

EADIE HEYDERMAN From the Department of Morbid Anatomy, St Thomas' Hospital Medical School, London SE] 7EH, UK The macroscopic and microscopic morphology of a Immunocytochemical methods are available for lesion is the basis of histopathological diagnosis. the demonstration of many of these substances in Conventional 'special' stains are ofvalue in diagnosis fixed tissue sections. The permanence of the reaction but they lack the specificity of immunological product together with the facility for simultaneous reagents. Thus the demonstration of immunoreactive pathological diagnosis make the immunoperoxidase calcitonin in a possible medullary carcinoma of the method the technique of choice in histopathology at thyroid is more useful than the demonstration of the present time (De Lellis et al., 1979). argyrophilia by a Grimelius stain; similarly, an immunochemical stain for immunoglobulin in plasma cells or lymphomas of B-cell lineage is more Methods (Fig. 1) conclusive than the pyroninophilia of ribosomal RNA shown by methyl green pyronin. For precise The immunoperoxidase technique is an immuno- pathological classification we may need to use logical method which may be used for the demon- functional criteria in addition to morphology. Many stration of various substances in tissue sections and products are readily demonstrable in routinely utilises labelled or unlabelled antibodies and the

cell copyright. processed and fixed surgical biopsies, and in material very stable enzyme, horseradish peroxidase. The obtained at necropsy, as well as in cryostat sections, most widely used substrate, diaminobenzidine, smears, monolayer cell cultures, and resin-embedded polymerises in the presence of peroxidase and tissue. The localisation of such cell products has hydrogen peroxide to form an insoluble brown assumed a role of growing importance in routine polymer which is deposited at the site of antigen- diagnostic histopathology as well as in research. antibody reaction. Lifting and subsequent loss of tissue sections may be a problem in immuno-

Applications cytochemical studies, especially when multistep http://jcp.bmj.com/ methods are used. It is important to cut the sections Tumours produce a wide array of appropriate and with a sharp microtome blade and to use a good inappropriate products including hormones, adhesive such as egg albumin; gelatin in the water- immunoglobulins, oncofetal antigens, and milk bath is frequently inadequate. The slides are best proteins. The localisation of such substances may be dried slowly overnight in a 56°C oven. of value in diagnosis (Heyderman, 1979), in suggest- ing a possible primary site for a tumour presenting 1 DIRECT METHOD on September 30, 2021 by guest. Protected with metastatic disease, in prognosis, and in suggest- The primary antiserum directed against the antigen ing the most suitable circulating marker to be used to be demonstrated is directly labelled with peroxi- for individual patient monitoring. Immunocyto- dase. This method is suitable only where antisera chemical studies are essential features of the are available in reasonable quantity for labelling diagnosis of renal disease (Zollinger and Mihatsch, and as a one-step method is the shortest. It is, 1978) and of some dermatological disorders however, probably less sensitive than the multiple (Cooperative Study, 1975). Virus particles (Huang, step methods. 1975) and other microorganisms may be visualised by immunocytochemistry not only in their character- istic loci but in previously unsuspected sites, and 2 INDIRECT (SANDWICH) METHOD abnormal storage products such as ai-antitrypsin (Table) (Figs 2 to 7) (Blenkinsopp and Haffenden, 1977) may be Here the first antibody is unlabelled and the second demonstrated. antibody, which is directed against the immuno- globulin of the species in which the first antibody is Received for publication 26 April 1979 raised, is conjugated to peroxidase (Nakane and 971 J Clin Pathol: first published as 10.1136/jcp.32.10.971 on 1 October 1979. Downloaded from

972 Eadie Heyderman

INSOLUBLE BROWN POLYMER IF tXYZDAB

DAB _T

DIRECT INDIRECT PAP ENZYME BRIDGE LABELLED ANTIGEN METHOD METHOD METHOD METHOD METHOD copyright. Fig. 1 Immunoperoxidase methods. XYZ = antigen under test in tissue on microscope slide; HRPO = horseradish peroxidase; In = conjugate.

Table Immunoperoxidase technique for paraffin-embedded sections

* 1 Dewax through xylene and alcohols to water.

2 Bleach acid haematin with 7-5 % H2O2 in distilled water. Wash off with tap water. 5 min http://jcp.bmj.com/ 3 Inhibit endogenous peroxidase with 2-28 Y. periodic acid in distilled water. Wash off with tap water. 5 min t4 Block aldehyde groups with 0-02 % potassium borohydride in distilled water. Wash off with tap water. Wash off with PBS (pH 7-2) containing 0-02% sodium azide. 2 min 15 Incubate in moist chamber with 100 dl Ist antibody diluted in I % ovalbumin in PBS. Wash off with PBS. 30 min or longer 6 Agitate in PBS bath containing a few drops of detergent (I Y. BRIJ 96). (Sigma) 15 min 7 Incubate with 100 ,ul 2nd antibody (indirect conjugate) diluted in I % ovalbumin in PBS. Wash off with PBS. 30 min 8 Agitate in PBS bath containing few drops of detergent (BRIJ). 15 min 9 Incubate in diaminobenzidine (DAB) (10 mg DAB freshly dissolved in 10 ml 0 03 % H502 in PBS). Wash with tap water. 5 min 10 Counterstain in Mayer's haemalum; blue in lithium carbonate; dehydrate, clear, and mount in XAM (GURR) or Permount (Fisher). 5 min on September 30, 2021 by guest. Protected

*For resin-embedded sections remove plastic with saturated alcoholic sodium hydroxide (allowed to mature for five days). Wash off with absolute ethanol. Wash off with tap water. ±8 have not found that pretreatment with normal serum from the species of animal in which the second antibody (7) was raised improves the results (unpublished data). 'Conservation of precious antisera or use of low-titre antibodies may be achieved with overnight or 24-hour incubation.

Kawaoi, 1974). The indirect method has advantages 3 PAP (UNLABELLED ANTIBODY) METHOD over the direct method of increased sensitivity and The first and second antibodies are unlabelled, and the small number of peroxidase conjugates required- the third reagent is an immune complex of one for each species in which antisera are raised. peroxidase-anti-peroxidase precipitated at equival- Anti-species antibodies are more readily available ence (Sternberger et al., 1970). The second antibody in quantity and when affinity purified may be acts as a bridge between the first antibody and the labelled to give excellent results in terms of sensi- anti-peroxidase which is raised in the same species tivity and specificity (Heyderman, 1979). as the first antibody. J Clin Pathol: first published as 10.1136/jcp.32.10.971 on 1 October 1979. Downloaded from

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C s --' G. I -3s FIG. 6 FIG. 7 Examples of wax (Figs 2-5) and resin-embedded (Figs 6 and 7) sections stained by the indirect immunoperoxidase technique. Fig. 2 Malignant teratoma intermediate stained with antiserum to the specific subunit of HCG. The giant syncytial cell (right) and wall of a vascular channel (left) are positive. Note that endogenous peroxidase in the red cells in the vessel has been completely inhibited. x 80 Fig. 3 Lactational focus in needle biopsy of breast from postpartum patient with infiltrating carcinoma. Stained with antiserum to a lactalbumin. The lactating breast (right) is strongly positive while the milk protein is absent from the carcinoma (left). x 32 Fig. 4 Infiltrating ductal carcinoma of the breast stained with an antiserum to epithelial membrane antigen (EMA). The stain is mainly concentrated on the luminal membrane but there is some cytoplasmic staining. (Reprintedfrom Journal of Clinical Pathology, 32, 35-39 1979.) x 80 Fig. 5 Non-involved kidney lateral to hypernephroma stained with antiserum to EMA. Only the luminal membrane and casts of the distal tubules are positive. (Reprintedfrom Journal of Clinical Pathology, 32, 35-39, 1979.) x 80 Fig. 6 Resin-embedded I section of moderately differentiated adenocarcinoma of the colon stained for CEA. The luminal membrane of the malignant acini and contained shed cells and debris are positive. x 128 Fig. 7 Resin section ofnon-involved 'normal' mucosa overlying tumour shown in Figure 5. The luminal membrane of the non-neoplastic glands is strongly positive. x 128 J Clin Pathol: first published as 10.1136/jcp.32.10.971 on 1 October 1979. Downloaded from

974 Eadie Heyderman

4 ENZYME BRIDGE (TRIPLE SANDWICH) ENDOGENOUS PEROXIDASE METHOD Peroxidase and peroxidase-like enzymes are present Here the first, second, and anti-peroxidase antibodies in many normal and neoplastic tissues, including red are applied sequentially, followed by free peroxidase blood cells, white cells, and peroxisomes in the liver. (Mason et al., 1969). This is the most time-consuming Peroxidase activity has been shown to be particularly of the methods, and, having the most steps and high in some oestrogen-sensitive tissues (Desombre washes, tissue sections are more likely to be lost. et al., 1975). A number of methods have been described for 5 LABELLED ANTIGEN METHOD inhibiting endogenous peroxidase and avoiding An unlabelled antibody directed against the antigen false positives due to its presence. The periodic acid- to be demonstrated is applied to the section, borohydride method shown in the Table, steps 2-4, followed by the antigen which has itself been inhibits the endogenous enzyme even in eosinophils labelled with peroxidase (Mason and Sammons, (in which peroxidase is difficult to block) and results 1979). This method is suitable only for materials in the crisp definition typical of periodic acid available in quantity for purification and labelling. treated sections (Heyderman and Neville, 1977). Since periodic acid oxidises sugar molecules contain- 6 MIXED AGGLUTINATION ing vicinal diols, opening the ring structure between IMMUNOCYTOCHEMICAL (MAGIC) METHOD such hydroxyl groups, the possibility exists that This is not an immunoperoxidase technique but may carbohydrate determinants may be destroyed. In be mentioned here as a variant of method 5 (above). these cases, as in the localisation of blood group It is used for the demonstration of enzymes whose substances, preliminary experiments with and bioactivity is destroyed by fixation but whose anti- without inhibition need to be carried out (see genic determinants are retained and should yield a below), and it may be necessary to use an alternative high degree of specificity. The first anti-enzyme anti- method for blocking endogenous peroxidase body is applied followed by the enzyme and its (Heyderman and Neville, 1977). specific substrate (Wachsmuth, 1976). copyright. 7 LOCALISATION BY ENZYMES OTHER TRYPSINISATION THAN PEROXIDASE There has been much interest recently in the Alkaline phosphatase has been used among others enhancement of immunocytochemical staining by and may be employed in a double labelling technique pretreatment of paraffin-embedded formalin-fixed (Mason and Sammons, 1978). Either the two first sections with trypsin or other proteolytic enzymes antibodies are raised in different species and one (Huang et al., 1976), particularly for the demonstra- second is labelled with tion of immunoglobulins. The difficulty about the

anti-species antibody http://jcp.bmj.com/ peroxidase and the other with alkaline phosphatase, use of such enzymes is that the technique is fickle so or the antibodies may be directly labelled. Other that not only is it easy to undertreat or overtreat but enzymes appear to be less suitable. the decision on the 'correct' treatment is made on what appears to be purely subjective grounds. The two most commonly used methods at the Furthermore, the possibility that the proteolytic present time are methods 2 and 3, the indirect or cleavage of tissue antigens may yield fragments sandwich technique and the PAP method. The common to many determinants is very real. At the latter is used in most laboratories largely because present time a better fixative such as Bouin's or on September 30, 2021 by guest. Protected reasonable reagents are available commercially, Zenker's is preferable and yields excellent results but, using such commercial reagents and comparing with most antigens including immunoglobulins, them with affinity-purified peroxidase-labelled without the need for trypsinisation (Heyderman and second antibodies, I have consistently failed to Monaghan, 1979; and unpublished observations). show increased sensitivity without loss of specificity. If trypsinisation is used, and various times of In addition, the PAP technique is one stage longer incubation and temperature, an absorption control (Heyderman and Neville, 1977). at each stage is necessary. My own experience is Many products retain their antigenic determinants that the degree of discrimination between strongly after fixation in Bouin's, Zenker's, or formalin positive cells and stroma or overlying epithelium is solutions, and wax or resin embedding and removal markedly reduced. (Heyderman and Monaghan, 1979). The immuno- peroxidase method is equally suitable for cryostat CARCINOGENICITY sections, smears, and monolayer tissue cultures. All the chromogenic substrates in present use are These may be grown on glass cover slips for staining best regarded as potentially carcinogenic or toxic or fixed as pellets and routinely embedded and cut. and should be treated with extreme caution. They J Clin Pathol: first published as 10.1136/jcp.32.10.971 on 1 October 1979. Downloaded from

Immunoperoxidase technique in histopathology: applications, methods, and controls 975 should be handled with gloves in a fume cupboard. negative control than normal non-immune serum Manufacturers should be encouraged to market the (see below). As purification of the antigen proceeds, substrates in convenient injection-type vials so that controls can be made more specific. This is not to no weighing or handling of the dry powder is suggest that the practice of absorbing an antiserum required. The formation of an aerosol during with impure antigen used to raise it is desirable. It weighing is a particular problem with diamino- could lead to a misleading impression of specificity. benzidine, which is still the best peroxidase substrate Controls are a progressive process, specificity for both light and electron microscopy. Fortunately, controls becoming more stringent and valuable as it is already obtainable ready weighed (BDH, Poole, chemical purification of the antigen proceeds. UK; Polysciences, USA). Absorption controls using, for example, absorption with whole serum as a control for one circulating Controls plasma protein are obviously of very limited value. There is little technical difficulty in staining sections using the immunoperoxidase technique. The 2 OTHER CONTROLS problems are the production or acquisition of good antisera and the validation of the results achieved (a) DAB alone with them. All too often submitted papers show If endogenous peroxidase is successfully inhibited only that the investigator has used differently (chronic myeloid leukaemic blood smears are useful labelled bottles of antiserum but has offered no test slides) the use ofsubstrate alone is an unnecessary evidence that the material demonstrated is really the control unless one is testing a new antibody directed material under test and not some irrelevant antigen against an antigen which could be sensitive to the revealed by an unwanted contaminating antibody in periodic acid used for inhibition. In preliminary one of the reagents. work on the localisation of blood group substance Monoclonal antibodies produced by hybridomas 'A', using an antiserum raised in rabbits by

(Kdhler and Milstein, 1976) will have the advantage Professor Lemieux (Lemieux, 1979) to synthetic copyright. of being well characterised and eventually available 'A' oligosaccharide, inhibition of endogenous in bulk so that laboratories world-wide can compare peroxidase was omitted until it could be shown results, knowing that the reagents are recognising that the antigenic determinants were insensitive to the same antigenic determinants. Until these periodate oxidation (Neville and Heyderman, 1979). reagents are in general use, careful controls in each Interpretation of positivity found both using sub- laboratory, regardless of the source of the antisera, strate alone and with the whole immunochemical are essential. sequence is difficult. Thus, in the case of the localisation of blood group substances, positive http://jcp.bmj.com/ 1 ABSORPTION CONTROLS staining could be due both to endogenous peroxi- The abolition of positive staining after absorption of dase and to the exogenous peroxidase of the the antibody with the antigen under test is the most second antibody peroxidase conjugate, binding to useful specificity control. This can be carried out specific anti 'A' serum applied to 'A' red blood cells. either by direct addition ofthe antigen and incubation Having inhibited endogenous peroxidase, positive overnight or better by incubation with antigen staining of the red cells could now be interpreted as coupled to agarose or insolubilised with gluta- due to the presence of 'A' substance. This was on September 30, 2021 by guest. Protected raldehyde (Avrameas and Ternyck, 1969), so confirmed by absorption of the antiserum with the avoiding production of soluble immune complexes synthetic oligosaccharide and consequent loss of which could bind to any Fc receptors in the tissue staining. section not destroyed by fixation. Confirmation of the specificity of this loss of activity may be obtained (b) Omission of one or more reagents or substitution by incubation of the antibody with an unrelated with buffer antigen (inappropriate antigen control) and of These are unconvincing controls and should be unrelated non cross-reacting antibodies with the required only in very preliminary experiments. antigen (inappropriate antibody absorption control). Absence of anti-human activity in second or These are the controls of choice. Where the antigen subsequent antibodies is best visualised using well- is not yet completely characterised (Heyderman characterised specific first antibodies on tissues et al., 1979) the immunogen or putative antigen containing previously demonstrated antigens. If may be used for an absorption control. If, as outlined indirect conjugates are used alone the section should above, it is shown only to absorb out the activity of first be washed in a protein solution (1 % ovalbumin the antiserum under test, it is a more powerful in PBS) to prevent non-specific binding. J Clin Pathol: first published as 10.1136/jcp.32.10.971 on 1 October 1979. Downloaded from

976 Eadie Heyderman (c) Non-immune serum produced with antisera raised in two different species The use of pre-immune bleeds or unrelated non- has the same distribution both antisera are directed immune sera of the same species as the specific against the same antigen (see (d) above). Further- antiserum is hallowed by tradition but is an in- more, a blocking system using an antiserum raised in adequate negative control. The essence of immuno- another species is an unsatisfactory negative control. cytochemical controls is the demonstration of The rationale of the system is that if an antiserum specificity. If the non-immune serum is negative and raised in a goat to the antigen under test is applied the specific antiserum positive, the only justifiable first, it will bind to all the available sites. If then a conclusions are that they are different, that rabbit antiserum to the same antigen is applied it will endogenous peroxidase has been inhibited, and not bind, and staining using anti-rabbit conjugate or that there are no unsuspected anti-human antibodies the PAP sequence will be negative (see Fig. 1). in the second or subsequent antisera. Absorption There are three possible objections: (a) Unless the controls are necessary to show that the animal into specificity of one of the antisera is established by an which the immunogen has been injected has 'seen' absorption control ((1) above), both could be only the antigen under test and not an impurity, or directed at a common contaminant. (b) Even if both that it does not already have antibodies that cross- antisera are directed at the same antigen one could react with human tissues. Non-immune sera may be directed at a carbohydrate determinant and the have interesting specificities for human tissues but other against a protein moiety of the same molecule, these are irrelevant to the validation of specificity of as with CEA (Ormerod, 1978). In this case the the antiserum under test. If, however, pre-immune antisera raised in different species do not block each sera stain human tissues, and this activity cannot be other since they bind to different parts of the removed by absorption with insolubilised tissue molecule (unpublished observation). (c) Antisera extracts, the animal is unsuitable for raising antisera raised in animals contain a population of antibodies for immunocytochemistry. The testing of pre-immune of differing specificities, binding constants, and bleeds before immunisation would avoid the use of immunoglobulin subtypes. Differences of this type such animals and waste of valuable antigens. could make interpretation difficult. Relatively small copyright. (d) Comparison with other antisera quantities of 'pure' antigen are required to make an Differences in the distribution of staining with two agarose column for absorption controls and the different antisera are evidence of differing specificity, antibody can be eluted and the column used again but similarity per se cannot be used as evidence of and again. Absorption controls remain the method identity. Cells secrete and store a variety of of choice. substances which can be localised morphologically (f) Radioimmunoassay and immunodiffusion data in only a small range of sites. Thus, antisera to a http://jcp.bmj.com/ number of membrane antigens may show similar While loss of activity after absorption of specific localisation (Heyderman and Monaghan, 1979), but antibody with antigen in either radioimmunoassay differences in specificity may be shown by examining (RIA) or immunodiffusion systems is reassuring, it other tissues, normal and neoplastic. is not conclusive. The results must be negative An epithelial membrane antigen (EMA) within the system and within the limits of sensitivity (Heyderman et al., 1979) and Pi pregnancy specific of the system under test-the immunoperoxidase glycoprotein (SP1) (Bohn, 1972) have the same system. membrane distribution in carcinoma of the colon, Provided pure label is used and cross-reacting on September 30, 2021 by guest. Protected but in the placenta EMA is present on the membrane antibodies are not present, RIA can be carried out in of the fetal villi, while SP1 is present in the syncytio- the presence of 'irrelevant' antibodies even though trophoblast (Horne et al., 1977). The fact that these same antibodies would stain tissue sections similar results are obtained with antisera from with their multiplicity of antigens. Similarly, in the various sources is not in itself a control. Not screening of antisera, non-precipitating or very low infrequently the immunogens come from the same titre antibodies may be undetectable by immuno- primary source or manufacturer, and contaminants diffusion but be quite suitable for immunocyto- that co-purify are likely to be problems in all chemistry (Heyderman et al., 1979). laboratories (Moss et al., 1978). It is necessary to Most commercial antisera are developed for RIA exercise constant vigilance, and as these problems or for immunodiffusion. It is rarely economically are reported in the literature suitable controls must feasible for manufacturers to affinity purify their be added. antisera. Though the label on the vial may be taken to indicate that the contained antiserum has activity (e) Blocking with antiserum raised in another species against the designated antigen, it cannot be taken to It is not possible to say that because staining indicate that other antibodies are not present. J Clin Pathol: first published as 10.1136/jcp.32.10.971 on 1 October 1979. Downloaded from

Immunoperoxidase technique in histopathology: applications, methods, and controls 977 Claims of the specificity of antisera based on the of proteins with glutaraldehyde and its use for the difference of distribution of staining, the fact that preparation of immunoadsorbents. Immunochemistry, the antisera came from several commercial differently 6, 53-66. labelled containers, and that normal non-immune Blenkinsopp, W. K., and Haffenden, G. P. (1977). Alpha-1-antitrypsin bodies in the liver. Journal of serum produces no staining must be viewed with Clinical Pathology, 30, 132-137. extreme caution if not scepticism. Bohn, H. (1972). Isolierung und Charakterisierung des schwanger-schafts-spezifischen P1-Glykoproteins. Blut, 3 POSITIVE CONTROLS 24, 292-302. It is useful to have a 'library' of normal and neo- Cooperative Study (1975). Uses for immunofluorescence plastic tissues to use as both positive and negative tests of skin and sera. Archives of Dermatology, 111, controls and to establish differences in antisera by 371-381. virtue of differences in staining. In studies on the DeLellis, R. A., Stemberger, L. A., Mann, R. B., Banks, localisation of a variety of tumour products, I test P. M., and Nakane, P. K. (1979). Immunoperoxidase new antisera on breast, colorectal, renal, bronchial, technics in diagnostic pathology. American Journal of Clinical Pathology, 71, 483-488. and lymphoid tumours as well as on placenta and Desombre, E. R., Anderson, W. A., and Kang, Y. H. pituitary. All the tumours are selected to include (1975). Identification, subcellular localization, and non-involved 'normal' tissue. Once the presence ofan estrogen regulation of peroxidase in 7,12-dimethylbenz antigen in a tissue has been established using (A) anthracene-induced rat mammary tumours. appropriate absorption controls, a section of the Cancer Research, 35, 172-179. known positive tissue should be included in each Heyderman, E. (1979). Immunocytochemistry in cancer batch of slides to ensure that absence of positive diagnosis. The Tenth Pfizer International Symposium. staining is not due to faulty technique. Cancer Assessment and Monitoring. Churchill However, since tumours frequently arise in an Livingstone, Edinburgh. (In press.) Heyderman, E., and Monaghan, P. (1979). Immuno- area of field change, claims for the localisation of peroxidase reactions in resin embedded sections. ectopic or inappropriate products using such Journal ofInvestigative and Cell Biology, 2, 119-122. 'normal' tissues or material from necropsies may be Heyderman, E., and Neville, A. M. (1977). A shorter copyright. difficult to interpret. For such studies previously immunoperoxidase technique for the demonstration of normal tissues, blocked in small thin portions and carcinoembryonic antigen and other cell products. obtained from traumatic or reconstructive surgery, Journal of Clinical Pathology, 30, 138-140. are required. Where death is the terminal event in a Heyderman, E., Steele, K., and Ormerod, M. G. (1979). protracted illness, anoxia, malnutrition, or cachexia A new antigen on the epithelial membrane: its immuno- could cause biochemical changes and postmortem peroxidase localisation in normal and neoplastic autolysis reflected in the apparent localisation of tissue. Journal of Clinical Pathology, 32, 35-39.

Home, C. H. W., Towler, C. M., and Milne, G. D. http://jcp.bmj.com/ abnormal products. (1977). Detection of pregnancy specific ,B-glycoprotein Immunocytochemical methods are valuable tools in formalin-fixed tissues. Journal of Clinical Pathology, in both routine histopathology and research. 30, 19-23. Meticulous techniques are important, as is critical Huang, S. N. (1975). Structural and immunoreactive appraisal of the results achieved. The discrimination characteristics of hepatitis B core antigen. American should be such that a slide may be read easily by a Journal of the Medical Sciences, 270, 131-139. novice, and the necessity for 'interpretation' of Huang, S. N., Minassian, H., and More, J. D. (1976). results usually implies unacceptable stromal staining. Application of immunofluorescent staining on paraffin on September 30, 2021 by guest. Protected The use of controls is essential but such controls sections improved by trypsin digestion. Laboratory need to be regularly reviewed as new possible Investigation, 35, 383-390. Kohler, G., and Milstein, C. (1976). Derivation of problems are reported in the literature. specific antibody-producing tissue culture and tumor lines by cell fusion. European Journal of Immunology, I thank Dr Saul W. Rosen and Dr Alan Rabson for 6, 511-519. their most useful criticism and discussion; Professor Lemieux, R. U. (1978). Human blood groups and carbo- Neville for encouragement; my former colleagues at hydrate chemistry. Chemical Society Reviews, 7, The Ludwig Institute of Cancer Research who were 423-425. involved in the preparation of many of the reagents Mason, D. Y., and Sammons, R. E. (1978). Alkaline and tissues used in these studies; and Ms Peggy phosphatase and peroxidase for double immuno- Jacobs for expert secretarial assistance. enzymatic labelling of cellular constituents. Journal of Clinical Pathology, 31, 454-460. Mason, D. Y., and Sammons, R. E. (1979). The labelled References antigen method of immunoenzymatic staining. Journal of Histochemistry and Cytochemistry, 27, 832-840. Avrameas, S., and Ternyck, T. (1969). The cross-linking Mason, T., Phifer, R., Spicer, S. S., Swallow, R. A., and J Clin Pathol: first published as 10.1136/jcp.32.10.971 on 1 October 1979. Downloaded from

978 Eadie Hevdermai Dreskin, R. B. (1969). An immunoglobulin-enzyme Immunology, 8, Supplement 8, 433-438. bridge method for localizing tissue antigens. Journal of Sternberger, L. A., Hardy, P. H., Jr., Cuculis, J. J., and Histochemistry and Cytochemistry, 17, 563-569. Meyer, H. G. (1970). The unlabeled antibody enzyme Mloss, J., Ross, P. S., Agosto, G., Birken, S., Canfield, method of immunohistochemistry. Preparation and R. E., and Vaughan, M. (1978). Mechanism of action properties of soluble antigen-antibody complex of choleragen and the glycopeptide hormones: is the (horseradish peroxidase-antihorseradish peroxidase) nicotinamide adenine dinucleotide glycohydrolase and its use in identification of spirochetes. Journal of activity observed in purified hormone preparations Histochemistry and Cytochemistry, 18, 315-333. intrinsic to the hormone? Endocrinology, 102, 415-419. Wachsmuth, E. D. (1976). The localisation of enzymes in Nakane, P. K., and Kawaoi, A. (1974). Peroxidase- tissue sections by immunohistochemistry. Conventional labelled antibody. A new method of conjugation. antibody and mixed aggregation techniques. Histo- Journal of Histochemistry and Cytochemistry, 22, chemical Journal, 8, 253-270. 1084-1091. Zollinger, H. U., and Mihatsch, M. J. (1978). Renal Neville, A. M., and Heyderman, E. (1979). Tumour Pathology in Biopsy. Springer, Berlin and New York. markers in human breast cancer. Tumour Markers. Elsevier, North Holland, Amsterdam. (In press.) Requests for reprints to: Dr E. Heyderman, Department Ormerod, M. G. (1978). Antigenic determinants of of Morbid Anatomy, St Thomas' Hospital Medical carcinoembryonic antigen. Scandinavian Journal of School, London SEI 7EH, UK. copyright. http://jcp.bmj.com/ on September 30, 2021 by guest. Protected