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made with the document.
See also: Tissue Stains: 49,
51, 52,
55, 56,
69, 72,
81.
Spills and disposal: 43,
116, 220,
253.
PRINCIPLE OF THE TEST.
Survival of tissue antigens for immunochemical staining
requires appropriate fixation.
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.
Formalin.
Ethanol.
B-5 fixative.
Bouin's solution.
STEP-BY-STEP DESCRIPTION.
1. SPECIMEN PREPARATION
1.1. Survival of tissue antigens for immunochemical staining may
depend on the type and concentration of fixative, on fixation time, and
on the size of the tissue specimen to be fixed. The SPECIAL
INSTRUCTIONS included in all kits should be consulted, to ascertain what
type of fixation is likely to give the best staining of the target antigen.
In general, increasingly weaker staining will be obtained when changing from
frozen tissue to tissue fixed in ethanol, B-5, Bouin's, and finally,
formalin.
1.2. Although 10% neutral phosphate-buffered formalin is generally
recommended as the best fixative for routine use, most kits have
been optimized to stain paraffin embedded sections which were
fixed in a wide range of fixatives. Zenker's fluid, B-5 (formol
sublimate) and Bouin's have often been recommended as milder
fixatives, especially for immunoglobulin.
1.3. The staining of surface antigens becomes increasingly weaker
when changing from unfixed specimen, e.g., cryostats, smears,
cytospins, to specimen fixed in acetone, to methanol/acetone, to
buffered formol acetone. However, morphology is best preserved
in the reverse order.
PARAFFIN EMBEDDED TISSUE.
2. TISSUE FIXATION FORMALDEHYDE-BASED SO~LUTIONS.
2.1 For optimal fixation in neutral buffered formalin,
tissue should be cut in blocks of approximately 1.0 x 1.0 x0.5 cm,
and immediately placed in at least 10 ml
formalin per block. The tissue should not remain in
formalin for more than 24 hours, and not more than 3
hours in B-5.
2.2 After fixation, blocks should be rinsed with water and
placed in 70% Flex, until ready to complete processing.
Tissues fixed in mercuric-chloride-containing fixatives
(Zenker's, B-5, etc.) should be
"de-Zenkerized" prior to application of hydrogen peroxide.
2.3. Processing may be completed using an automatic tissue
processor, graded alcohol substitute, xylene substitute,
and paraffin wax.
Paraplast and other plastic containing compounds may
reduce staining if incompletely removed. Temperatures
greater than 60o C MUST be avoided; rapid high
temperature processing destroys antigenicity.
2.4. Formaldehyde is known to induce stearic changes in
antigens by forming intermolecular cross linkages.
These modifications can only partly be compensated by
greater staining sensitivities. In particular,
cell surface antigens tend to be more denatured during
fixation with formaldehyde-based solutions than
cytoplasmic antigens.
3. TISSUE FIXATION IN ETHANOL (FLEX)
3.1. One recommended procedure for fixation in ethanol is to
incubate the tissue-blocks in absolute alcohol for 48
hours at room temperature, followed by two 1-hour
incubations each in fresh xylene substitutte, and a 2-hour bath in
liquid paraffin. The tissue may then be placed in
cassettes and embedded in paraffin.
4. REMOVAL OF PARAFFIN AND DEHYDRATION OF TISSUE
4.1. It is very important that the embedding medium be
completely removed from the specimen. Any residual
medium will cause an increase in staining. For this
reason, all xylene substitute and Flex solutions should be
changed in place of xylene with equal results.
4.1.1. Place section in 56-60o C oven for 3 minutes.
(Caution: Oven temperature must not exceed 60o C).
4.1.2.
(b) Transfer slide immediately into xylene bath,
and incubate for three minutes. Repeat for three minutes.
Repeat once.
4.1.3.
Drain off excess liquid and place slide in
fresh absolute ethanol for 3 minutes. Repeat once.
4.1.4. Drain off excess liquid and place slide in 95%
ethanol for 3 minutes. Repeat once.
4.1.5. Rinse slide in gently running water for 30 seconds.
Avoid a direct jet, which may wash off the
loosen the tissue-section.
Commence staining procedure.
5. FROZEN SECTION
5.1.
Cryostat sections (5-8 microns)
should be cut from snap frozen blocks
(approximately 1.0 x 1.0 x 0.5 cm),
and air dried for 2-24 hours,
or in a freeze-drying apparatus,
if available, and processed immediately.
Sections (or tissue) may also be wrapped,
air-tightened stored at -20o C or lower.
After storage, sections should be
brought to room temperature before unwrapping,
and fixed in acetone for 10 minute,
or in buffered formol acetone for 10 minutes,
or in buffered formol acetone for 30 seconds.
Allow sections to air dry.
After submerging the slides
in Tris buffer for 5 minutes,
commence staining procedure.
5.2.
When performing the PAP procedure, endogenous
peroxidase activity can be suppressed, if necessary,
by fixing the slide for 20 minutes in freshly prepared
methanolic hydrogen peroxide (50 ml ~water,~ plus 200 ml
absolute methanol), followed by a brief rinse under tap
water. Place the slides in a Tris buffer bath for five minutes,
and commence staining procedure.
Note, however, that methanolic hydrogen
peroxide may destroy antigenicity of surface proteins
and detach frozen sections from glass. No suppression
of peroxidase activity is necessary when following the
this procedure.
6. OTHER SPECIMENS
6.1. Cytospins and imprints.
6.2.
Smears may be dried in air for 2-24 hours and
processed for staining immediately or wrapped
airtight and stored at -20o C or lower.
Air dried or slowly-thawed specimens
may then be fixed for one minute
in acetone or for 30 seconds in buffered
formol acetone. Fixation in acetone-methanol
(1:1), or acetone-methanol-formalin (10:10:1) for
1 minute is acceptable also. In general, buffered
formol acetone will give good preservation of cell
morphology while still retaining immunoreactivity
of most surface antigens.
6.3. As for frozen sections, endogenous peroxidase activity can be suppressed
by fixation in methanolic hydrogen peroxide for 20 minutes.
6.4. Following a 5 minute bath in Tris buffer, staining can be commenced.
REFERENCES.
1. Hopwood D.
Fixation and Fixatives. Chapter 3. pp. 63-84.
In: Bancroft JD, Gamble M.
Theory and Practice of Histological Techniques. Fifth Edition.
Edinburgh: Churchill Livingstone. 2002;:63-84.
ISBN 0-443-06435-0, 796 pages.
2. Lillie RD, Fulmer HM.
Histopathological Technique and Practical Histochemistry. Fourth Edition.
New York: McGraw-Hill. 1976;:.
3. Medawar PB.
The rate of penetration of fixatives.
J Royal Micro Soc. 1941;61:46-57.
4. Walker JF.
Formaldehyde.
London: Chapman & Hall. 1964;:.
5. Pearse AGE.
Histochenmistry Theoretical and Applied. Fourth Edition.
Edinburgh: Churchill Livingstone. 1980;1:.
6. Trump BF, Ericsson JLE.
The effect of the fixative solution on the ultrastructure of cells
and tissues. A comparative analysis with particula attention to the
proximal convoluted tubule of the rat kidney.
Lab Invest. 1965;14:1245-1325.
7. 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.
8. 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.