J Clin Pathol: first published as 10.1136/jcp.37.10.1190 on 1 October 1984. Downloaded from

1190 Technical methods Phosphate buffer at pH 8*0 gave the sharpest 2 Rozenszajn L, Leibovich M, Shoham D, Epstein J. The esterase staining reactions, although there was little differ- activity in megaloblasts, leukaemic and normal haemopoietic cells. Br J Haematol 1968; 14:605-19. ence at pH 7-0 or pH 7-5. As the buffer pH was 3Hayhoe FGJ, Quaglino D. Haematological cytochemistry. Edin- increased above pH 8-0 staining with both substrates burgh: Churchill Livingstone, 1980. became progressively weaker, especially above pH 4Li CY, Lam KW, Yam LT. Esterases in human leucocytes. J 9.0. Below pH 7-0 staining with a-naphthyl butyrate Histochem Cytochem 1973;21:1-12. Yam LT, Li CY, Crosby WH. Cytochemical identification of became weaker, and below pH 5*0 staining with monocytes and granulocytes. Am J Clin Pathol 1971;55:283- naphthol AS-D chloroacetate began to disappear. 90. 6 Armitage RJ, Linch DC, Worman CP, Cawley JC. The morphol- This work was supported by a Medical Research ogy and cytochemistry of human T-cell subpopulations defined by monoclonal antibodies and Fc receptors. Br J Haematol Council project grant. I thank Professor FGJ 1983;51:605-13. Hayhoe for valuable advice. References Requests for reprints to: Dr DM Swirsky, Department of Gomori G. Chloroacyl esters as histochemical substrates. J His- Haematological Medicine, University Clinical School, Hills tochem Cytochem 1953;1:469-70. Road, Cambridge CB2 2QL, England.

Simple technique to identify identification of the two compounds on the same haemosiderin in slide. immunoperoxidase stained Material and methods

Sections were cut from biopsy specimens of surgi-copyright. sections cally removed pituitary gland which had been embedded in paraffin and fixed in formalin. Ten CAROLE D NICKOLS Department of Morbid 3 ,tm serial sections were prepared and dried in a Anatomy. The London Hospital, Whitechapel, Lon- 37°C incubator for 12 h. don El IBB The immunoperoxidase staining method used was an adaptation of the method of Kovacs et al.' The Identifying iron compounds in immunoperoxidase method differed from the original technique by stained sections usually presents little or no problem using antihuman prolactin antiserum (Mercia http://jcp.bmj.com/ to the trained eye. The distinction between peroxi- Brocades) at a dilution of 1/1000 (diluent 0-15 M dase positive staining and haemosiderin becomes phosphate buffered saline, pH 7.2) at 4°C for 1 h on important when one is in doubt as to which com- material obtained from surgical biopsies. Before pound is giving the "brown positive" result. staining, the natural endogenous peroxidase activity Recent biopsies have presented this problem. was blocked using the modified technique that Although naturally occurring endogenous peroxi- Slocombe et a12 used in the demonstration of blood dase activity has been blocked in the sections there group antigens. has been confusion between the brown staining of After treatment with 1% osmium, the last stage of on September 27, 2021 by guest. Protected haemosiderin and the positive brown staining of the the original technique before mounting the section, 3-3' diaminobenzidine tetrahydrochloride, es- the sections were gently washed in running tap water pecially in weakly positive sections. for 5 min. They were then washed several times in The staining of serial sections with haematoxylin distilled water. The original technique of Perls3- and eosin and by Perls' Prussian blue is routinely heated potassium ferrocyanide and 1 % hydrochloric performed when this problem arises. But unless a acid-was then applied to show the haemosiderin in comparator microscope is used to check the identity the section. of the stained cell, the problem remains. Counter- The Perls' Prussian blue and the uncounterstained staining the peroxidase stained slide with Perls' peroxidase serial slides were compared with the Prussian blue technique, however, permits the peroxidase section counterstained with Perls' Prus- sian blue using a Zeiss comparison bridge mounted on two Zeiss Standard 18 microscopes. (Carl Zeiss Accepted for publication 14 May 1984 (Oberkochen) Limited) J Clin Pathol: first published as 10.1136/jcp.37.10.1190 on 1 October 1984. Downloaded from Technical methods 1191 Results It has previously been used successfully after the peroxidase-antiperoxidase method of Sternberger The three slides of the serial sectioned material- and Cucullis4 in renal cell carcinoma,5 where large peroxidase uncounterstained, Perls' Prussian blue, amounts of iron pigment were present in the section. and the peroxidase counterstained with Perls' Prus- With the increased use of immunoperoxidase sian blue-were carefully compared by matching the techniques in surgical pathology,6 the addition of architecture of the fields using a comparison bridge. this simple but old technique to the modern one Neither the prolactin nor the haemosiderin compo- could further increase its specificity. nents seemed to be diminished as a result of the extra staining procedure. I thank Mr Ken Swettenham and Professor Berry Low background staining resulted from the use of for encouragement and help with this paper. reasonably fresh potassium ferrocyanide crystals; the background staining appeared to increase in References proportion to the increased shelf life of the crystals, 'Kovacs K, Corenblum B, Sirek AMT, Penz G, Ezrin C. Localiza- although this was not measured histoquantitatively. tion of prolactin in chromophobe pituitary adenomas: study of But distinction could still be made between the necropsy material by immunoperoxidase technique. J Clin peroxidase positive staining and the Pathol 1976;29:250-8. haemosiderin. 2 Slocombe GW, Berry CL, Swettenham KV. The variability of The increased time in washing after the osmium blood group antigens in gastric carcinoma as demonstrated by treatment, when performed with care, did not seem the immunoperoxidase technique. Virchows Arch (Pathol to increase the fragility of the sections, although Anat) 1980;387:289-300. more 3 Perls Von M. Nachweis von Eisenoxyd in gewissen Pigmenten. vigorous washing for longer periods was not Virchows Arch (Pathol Anat) 1867;39:42-8. attempted. 4 Sternberger LA, Cucullis JJ. Method for enzymatic intensification of immunocytochemical reaction without use of Discussion labelled antibodies. J Histochem Cytochem 1969; 17:190. Lindop GBM, Fleming S. Renin in renal cell carcinoma-an immunocytochemical study using antibody to pure human Hormone identification plays an important part in renin. J Clin Pathol 1984;37:27-31.

the diagnosis of pituitary adenomas. Often, biopsy 'Taylor CR, Kledzik G. Immunologic techniques in surgical copyright. specimens obtained from surgically removed pit- pathology-a spectrum of "new" special stains. Hum Pathol uitary gland contain sites of old haemorrhage which 1981; 12:590-6. are sparse, diffuse, and intermingled with the hor- mone producing cells. At the sites the differentiation Requests for reprints to: Miss CD Nickols, Department of between hormone and haemosiderin must be clear, Experimental Pathology, Institute of Pathology, The Lon- which is why the technique was originally evaluated. don Hospital, Whitechapel, London El 1BB, England. http://jcp.bmj.com/

expensive. A simple method of storing a tissue sam- Storage of skin biopsies at 70°C ple for subsequent culture would be useful in these for future fibroblast culture circumstances and would also have other practical uses in a clinical cytogenetics or tissue culture laboratory-for example, when samples are deli- KJ FOWLER Birth Defects Research Institute, Royal vered at on September 27, 2021 by guest. Protected Children's Hospital Research Foundation, inconvenient times. Parkville, Victoria Australia The cryoprotective effect of glycerol on frozen 3052, spermatozoa was reported in 1949.' This resulted in many techniques which describe the storage of fro- zen mammalian cells and tumours.24 In 1959, It is difficult to know when to establish fibroblastic dimethyl sulphoxide was used to prevent the cell cultures from paediatric necropsies. Newborn haemolysis of frozen red blood cells.5 Five years babies who seem to have defects due to chromo- later Lehr et al6 described the successful transplanta- some aneuploidy may die before the results of tion of skin autografts which had been previously lymohocytic karyotypes are known. Lysosomal stor- frozen using cryoprotective agents. age diseases requiring confirmation by enzyme assay We report here a simple method of storing skin on cultured cells may be suspected only when mic- biopsies (2-3 mm3) at -70°C in culture medium roscopical examination of tissues has been com- plus dimethyl sulphoxide for 15-23 days without pleted. Establishing a culture from every necropsy is loss of capacity for fibroblastic cell growth.