Skip to main content
Journal of Clinical Pathology logoLink to Journal of Clinical Pathology
. 1978 Oct;31(10):974–983. doi: 10.1136/jcp.31.10.974

Demonstration of immunoglobulin in cryostat and paraffin sections of human tonsil by immunofluorescence and immunoperoxidase techniques. Effects of processing on immunohistochemical performance of tissues and on the use of proteolytic enzymes to unmask antigens in sections.

R C Curran, J Gregory
PMCID: PMC1145462  PMID: 361759

Abstract

A fluorescein isothiocyanate (FITC) technique and one based on peroxidase-antiperoxidase (PAP) were used to study the distribution of immunoglobulin (Ig) in cryostat and paraffin sections of human tonsil. Trypsin and other proteolytic enzymes were used to 'unmask' the antigen in paraffin sections. The effects of processing, and particularly of fixation, on the immunohistochemical response of tissues were studied. The FITC and PAP methods detected Ig in paraffin and cryostat sections equally well. The distribution of the antigen was the same with both methods but the PAP method was the more informative. Formaldehyde-sucrose solution proved more suitable for fixing tissues for immunohistochemistry than glutaraldehyde. Trypsin revealed antigen in parraffin sections more efficiently than pepsin, papain, or pronase. Surface Ig (s-Ig) could be demonstrated in trypsinised paraffin sections but less effectively than in cryostat sections. Trypsinised paraffin sections were, however, more suitable for intracellular Ig (c-Ig) than cryostat sections although the performance of cryostat sections could be improved by prior fixation with a coagulative fixative.

Full text

PDF
974

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baklien K., Brandtzaeg P. Comparative mapping of the local distribution of immunoglobulin-containing cells in ulcerative colitis and Crohn's disease of the colon. Clin Exp Immunol. 1975 Nov;22(2):197–209. [PMC free article] [PubMed] [Google Scholar]
  2. Brandtzaeg P., Baklien K., Fausa O., Hoel P. S. Immunohistochemical characterization of local immunoglobulin formation in ulcerative colitis. Gastroenterology. 1974 Jun;66(6):1123–1136. [PubMed] [Google Scholar]
  3. Brandtzaeg P. Mucosal and glandular distribution of immunoglobulin components. Immunohistochemistry with a cold ethanol-fixation technique. Immunology. 1974 Jun;26(6):1101–1114. [PMC free article] [PubMed] [Google Scholar]
  4. Burns J., Hambridge M., Taylor C. R. Intracellular immunoglobulins. A comparative study on three standard tissue processing methods using horseradish peroxidase and fluorochrome conjugates. J Clin Pathol. 1974 Jul;27(7):548–557. doi: 10.1136/jcp.27.7.548. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Curran R. C., Gregory J. The unmasking of antigens in paraffin sections of tissue by trypsin. Experientia. 1977 Oct 15;33(10):1400–1401. doi: 10.1007/BF01920206. [DOI] [PubMed] [Google Scholar]
  6. Curran R. C., Jones E. L. Immunoglobulin-containing cells in human tonsils as demonstrated by immunohistochemistry. Clin Exp Immunol. 1977 Apr;28(1):103–115. [PMC free article] [PubMed] [Google Scholar]
  7. Denk H., Radaszkiewicz T., Weirich E. Pronase pretreatment of tissue sections enhances sensitivity of the unlabelled antibody-enzyme (PAP) technique. J Immunol Methods. 1977;15(2):163–167. doi: 10.1016/0022-1759(77)90027-8. [DOI] [PubMed] [Google Scholar]
  8. GRAHAM R. C., Jr, LUNDHOLM U., KARNOVSKY M. J. CYTOCHEMICAL DEMONSTRATION OF PEROXIDASE ACTIVITY WITH 3-AMINO-9-ETHYLCARBAZOLE. J Histochem Cytochem. 1965 Feb;13:150–152. doi: 10.1177/13.2.150. [DOI] [PubMed] [Google Scholar]
  9. Garvin A. J., Spicer S. S., Parmley R. T., Munster A. M. Immunohistochemical demonstration of IgG in Reed-Sternberg and other cells in Hodgkin's disease. J Exp Med. 1974 May 1;139(5):1077–1083. doi: 10.1084/jem.139.5.1077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jefferis R., Mathews J. B. Studies of human IgD myeloma proteins. Proteolytic digestion patterns. Immunochemistry. 1977 Mar;14(3):171–178. doi: 10.1016/0019-2791(77)90191-4. [DOI] [PubMed] [Google Scholar]
  11. Kuhlmann W. D., Avrameas S., Ternynck T. A comparative study for ultrastructural localization of intracellular immunoglobulins using peroxidase conjugates. J Immunol Methods. 1974 May;5(1):33–48. doi: 10.1016/0022-1759(74)90043-x. [DOI] [PubMed] [Google Scholar]
  12. Leduc E. H., Scott G. B., Avrameas S. Ultrastructural localization of intracellular immune globulins in plasma cells and lymphoblasts by enzyme-labeled antibodies. J Histochem Cytochem. 1969 Apr;17(4):211–224. doi: 10.1177/17.4.211. [DOI] [PubMed] [Google Scholar]
  13. Maunsbach A. B. The influence of different fixatives and fixation methods on the ultrastructure of rat kidney proximal tubule cells. I. Comparison of different perfusion fixation methods and of glutaraldehyde, formaldehyde and osmium tetroxide fixatives. J Ultrastruct Res. 1966 Jun;15(3):242–282. doi: 10.1016/s0022-5320(66)80109-0. [DOI] [PubMed] [Google Scholar]
  14. Mazurkiewicz J. E., Nakane P. K. Light and electron microscopic localization of antigens in tissues embedded in polyethylene glycol with a peroxidase labeled antibody method. J Histochem Cytochem. 1972 Dec;20(12):969–974. doi: 10.1177/20.12.969. [DOI] [PubMed] [Google Scholar]
  15. Nakane P. K. Recent progress in the peroxidase-labeled antibody method. Ann N Y Acad Sci. 1975 Jun 30;254:203–211. doi: 10.1111/j.1749-6632.1975.tb29170.x. [DOI] [PubMed] [Google Scholar]
  16. Pich A., Bussolati G., Carbonara A. Immunocytochemical detection of casein and casein-like proteins in human tissues. J Histochem Cytochem. 1976 Aug;24(8):940–947. doi: 10.1177/24.8.822100. [DOI] [PubMed] [Google Scholar]
  17. Reading M. A digestion technique for the reduction of background staining in the immunoperoxidase method. J Clin Pathol. 1977 Jan;30(1):88–90. doi: 10.1136/jcp.30.1.88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Vladutiu G. D., Bigazzi P. E., Rose N. R. Localization of a primate-specific esterase using immunofluorescence and immunoperoxidase techniques. J Histochem Cytochem. 1973 Jun;21(6):559–567. doi: 10.1177/21.6.559. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Pathology are provided here courtesy of BMJ Publishing Group

RESOURCES