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United States Government Work, uncopyrighted, public-domain,
DRAFT COPY ONLY. This document does not necessarily represent the views
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made with the document.
PRINCIPLE OF THE TEST.
To stain paraffinized tissue sections for immunologically stainable proteins,
required in the diagnosis of certain diseases encountered
in anatomic pathology.
SPECIMEN REQUIRED.
All human tissue excised at surgery, outpatient clinics, and postmortems,
fresh or in fixative, along with a filled-out Tissue Examination Form
(U. S. Standard Form 515, USSF515).
REAGENTS, INSTRUMENTATION.
Distilled water.
10% Neutral buffered formalin.
Paraffin sections at 6 microns.
Absolute ethanol.
Absolute methanol.
3% Hydrogen Peroxide (H2O2).
Undiluted NGS. (NGS-normal goat serum).
Primary antiserum.
Non-immune rabbit serum.
PBS buffer (pH 7.6).
Goat anti-rabbit IgG.
Rabbit PAP (1:80 dilution in PBS).
Freshly prepared solution of DAB (5% DAB in PBS buffer, pH 7.4).
Gill or Mayer Hematoxylin.
Bluing agent.
Xylene substitute.
STEP-BY-STEP DESCRIPTION.
1. The paraffin is removed from the tissue sections
by treatment with 3 changes of xylenes, 5 minutes each change.
2. Absolute ethanol, 2 changes for 1 minute each.
3. Following this, immerse in absolute methanol,
containing 3% Hydrogen Peroxide (H2O2),
for 15 - 30 minutes, to abolish endogenous peroxidase activity.
4. Then pass through a decreasing series of ethanol solutions to tap water.
5. Treat for 30 minutes with undiluted NGS. (NGS-normal goat serum).
6. Excess goat serum is shaken off, and the primary antiserum
is added to the sections.
7. As controls, the primary anti-serum is replaced with similar
dilutions of non-immune rabbit serum. Anti-serum dilutions and incubation
times can be found on following pages. Incubations are conducted at room
temperature.
8. Tissue sections are washed for 5 minutes in 2 changes of PBS buffer
(pH 7.6).
9. Excess buffer is shaken from the slides, and goat anti-rabbit IgG
(1:50 dilution in PBS) is added to the sections for 30 minutes.
10. Washed in PBS, 2 changes - 5 minutes each.
11. Rabbit PAP (1:80 dilution in PBS) added to the sections
for 30 minutes.
12. Wash in PB8, 2 changes - 5 minutes each.
13. Antibody localization in the tissue sections is detected
by placing them in a filtered, freshly prepared solution of DAB (5% DAB in
PBS buffer, pH 7.4, containing 1 ml of 3% Hydrogen Peroxide
(H2O2), for 5-10 minutes).
14. After treatment with DAB, the sections are rinsed
with PBS buffer, washed in tap water.
15. Stain with Gill or Mayer Hematoxylin for 2 - 3 minutes.
16. Wash in tap water for 1 minute, blue in bluing agent, wash in tap water.
17. Pass through an increasing series of ethanol solutions
into xylene substitute.
18. Coverslip. Positive area will be stained
brown; nuclei are blue.
REFERENCES.
1. Miller KD.
Immunohistochemical Techniques. Chapter 21. pp. 421-464.
In: Bancroft JD, Gamble M.
Theory and Practice of Histological Techniques. Fifth Edition.
Edinburgh: Churchill Livingstone. 2002;21:421-464.
ISBN 0-443-06435-0, 796 pages.
From: Table 20.1. Table of Clusters of Differentiation. pp. 442-444.
bcl2 ..... Follicular lymphoma.
CD2 ..... T cells.
CD3 ..... T cells.
CD4 ..... T-helper cells.
CD5 ..... T cells.
CD8 ..... T-suppressor cells.
CD15 ..... Hodgkin cells.
CD20 ..... B cells
CD21 ..... Follicular dendritic cells
CD23 ..... Lymphocytic lymphoma.
CD30 ..... Hodgkin cells.
CD56 ..... Small cell carcinoma.
CD68 ..... Monocytes, macrophages.
CD79a ..... B cells.
CD117 ..... Gastric stromal tumor.
Chromogranin ..... Carcinoid tumor.
CK 5/6 ..... Epithelioid mesothelioma.
CK 20 ..... Transitional cell carcinoma.
Cytokeratin ..... Adenocarcinoma.
Cytokeratin LP34 ..... Prostatic basal cells.
Desmin ..... muscle.
Epithelial membrane Antigen ..... Anaplastic large cell lymphoma.
GFAP ..... Astrocytoma.
IgA ..... B cells.
IgD ..... B cells.
IgG ..... B cells.
IgM ..... B cells.
S100 ..... Malignant melanoma.
Vimentin ..... Mesenchymal cells.
2. Krenacs L, Bagdi E, Krenacs T.
Immunocytopathology of Lymphomas.
In: loc. cit.
3. Barr N, Wu N, Taylor CR.
Immunocytopathology of Routine Histopathology. Chapter 24. pp. 537-552.
In: loc. cit.
4. Prophet EB, Mills B, Arrington JB, Sobin LH, eds.
Laboratory Methods in Histotechnology.
Washington, DC: Armed Forces Institute of Pathology. 1992;:.
ISBN 1-881041-00-X, 278 pages.
5. Mikel UV, ed.
Advanced Laboratory Methods in Histology and Pathology.
Washington, DC: Armed Forces Institute of Pathology. 1994;:.
ISBN 1-881041-13-1, 254 pages.