Immunocytochemistry of Frozen
and of Paraffin Tissue Sections
Immunocytochemistry of tissue sections can yield
valuable information as to the location of antigens.
Thus it can determine whether the antigen is ubiquitous
or is present only in certain tissues. It can further
determine whether all cells in a given tissue are positive
for a given antigen or whether the antigen is
restricted to one or a few specialized cell types within
the tissue. Its uses are not limited to normal tissues,
and testing of tumor tissues in immunocytochemistry
can yield information important for determination of
tumor type. As stated in the articles on immunocytochemistry
of cultured cells, choice of antibodies and of
fixation method can be of critical importance. New
antibodies should be tested on both frozen sections
and paraffin sections of a variety of tissues in which
the antigen is thought to be present. In general, more
antibodies will react on frozen sections than on paraffin
sections; however, morphology is better preserved
in the paraffin sections.
For other reviews of methods, see Denk (1987) and
Sternberger (1979), and for overviews of the use of
these methods in histopathology, see Osborn and
Weber (1983), Tubbs et al.
(1986), Jennette (1989), and
Osborn and Domagala (1997).
II. MATERIALS AND
See the article by Osborn in this volume. Establish
working dilutions by running a dilution series. Usually monoclonal antibodies can be diluted 1:5 to
1:30, ascites fluid 1 : 100 to 1 : 1000, and polyclonal antibodies
1:20 to 1:40, but the dilutions depend critically
on the detection technique that is employed. Dilute
both primary and secondary antibodies as far as possible
to save money and to avoid unspecific reactions.
Use a Leica CM1900 rapid sectioning cryostat
(www.leica-microsystems.com). A useful accessory is a
freezing head with variable temperature control. Use
a C-knife and resharpen when necessary.
OCT 4583 compound from Miles Laboratories.
D. Paraffin Embedding
Automatic machines are useful only for laboratories
that process a large number of samples. Check equipment
for paraffin embedding in the local pathology
department before purchasing.
E. Sliding Microtomes
These are relatively inexpensive and can be used
with disposable knives.
F. Water Baths for Histology
These are thermostatically controlled and relatively
shallow, e.g., 20cm in diameter and 4cm deep.
A microscope equipped with the appropriate filters
to view immunofluorescence specimens is important.
To view peroxidase- or streptavidin-biotin-stained
specimens only a simple light microscope is required.
A. Cryostat Sections
1. Freezing of Tissue Blocks
- Fill the inner beaker of the freezing apparatus
with isopentane and the outer beaker with liquid nitrogen
about 30min before freezing tissues (Fig. 1). Use
reagent-grade isopentane. Measure the temperature
and wait until isopentane reaches -120 to -130°C. At
-155°C the isopentane will freeze.
- Dissect tissues. Cut into small blocks (~4-7mm)
using a scalpel. Place block on a prenumbered square
of paper with the surface that will be sectioned furthest
away from the paper. For small specimens, e.g.,
vessels, place a drop of Tissue-tek on the paper and
then add the tissue.
- Drop tissue blocks into iospentane. Leave for at
least 30s. Remove blocks with plastic tweezers and
transfer directly to plastic vials (scintillation vials 6× 2.5 cm work well) or metal cans (~3 × 3 cm) with screw
tops that have been precooled on dry ice. Close vials and store in a -70°C freezer. Tissue blocks are stable
for several years.
|FIGURE 1 Apparatus used to freeze tissues.
2. Cutting Cryostat Sections
B. Paraffin Sections
1. Embedding Tissues in Paraffin
- Wash microscope slides by dipping in acetone,
air dry, and store at room temperature. Store plastic
tweezers and brush in the cryostat. Precool cryostat
and freezing head.
- Place Tissue-tek on precooled freezing head and
mount block so that the larger side of the cut section
is at 90° to the knife blade.
- The optimal cutting temperature is different for
different tissues. Most tissues cut well at -15 to -20°C. For liver, use -10°C. If the sections wrinkle as they are
cut and look mushy, decrease the temperature. If
the sections have cracks and look brittle, increase the
- To cut sections use the C-knife. First trim the
block to get a good cutting surface. Then adjust the
section thickness to 5µm and use the automatic
advance. Now cut three or four sections so that the
preset section thickness is achieved. Then bring the
antiroll plate on the knife to stop the section from
rolling up and to keep it fiat on the knife. The antiroll
plate must be parallel to the knife edge and should
only protrude very slightly over the edge.
- For optimal sections the knife and the antiroll
plate must be kept clean (use a soft cloth dipped in
acetone). Always clean the knife in the cutting direction
and never the reverse so as not to damage the
cutting edge of the knife.
- Remove the antiroll plate and hold the microscope
slide over but not touching the cut section. The
section should now spring on to the slide because of
the difference in temperature between knife and slide.
The quality of the section can be checked using toluidine
blue or hematoxylin-eosin staining (see later).
- Dry the sections at room temperature for 30min.
Then either use directly or place in a slide box and put
in a -70°C freezer. Cut sections are stable for months
or years at -70°C.
- Sections from a few tissues may not stick firmly
enough to slides and may come off during subsequent
processing. If this happens, try coating slides with
0.1% polylysine (e.g., Sigma Cat. No. 8920) in water.
Human material is often received from the clinic
already embedded in paraffin. Protocols vary depending on the clinic, with time of fixation in formaldehyde
being very variable (e.g., 4 h to over the weekend). To
embed animal tissue in the laboratory, cut into 4 to 7-
mm blocks and place it for 4-8 h in 3.7% formaldehyde
in phosphate-buffered saline (PBS), 1 h in 50% ethanol,
2 × 1 h in 70% ethanol, 2 × 1 h in 96% ethanol, 2 × 1 h
in 100% ethanol, 1 × 1 h in xylene, 1 × 2 h in xylene, and
2 × 2 h in Paraplast Plus (Shandon).
2. Cutting Paraffin Sections
|FIGURE 2 Correct adjustment of knife and of
cutting angle to cut
- To obtain very thin sections (1-2µm), put the
paraffin blocks in a freezer at -20°C for about 30min.
Mount the block in the holder of a sliding microtome.
If the block cuts well do not use the automatic advance,
but rely instead on the natural expansion of the block
as it warms up to advance the block. If the block is not
easy to cut, use the automatic advance set at a thickness
of 1-2 µm. Correct adjustment of the knife and of
the cutting angle is very important. Use an inclination
angle (β) of 15° (Fig. 2). If the inclination angle is less
than 10° the knife will not cut the block, and if it is
greater than 15° the block will break.
- Trim the block until the cutting surface is
optimal. Then cut a section, using a paint brush to
draw the section onto the knife so that it does not roll
- Dip a second paint brush in water so that the
section will adhere to it and move the section to a
water bath held at 40-45°C. If the section is placed with
the shiny smooth surface touching the water the
warmth will smooth out the section and wrinkles will
- Place a microscope slide under the section and,
using a brush, position the section on the slide.
- Dry the sections. For immunohistochemical
methods, dry overnight at 37°C. For normal histological methods, set drying oven to 60°C so that the
paraffin melts in part during the drying step and dry
for 1-2 h.
Immerse sections 2 × 10min in xylene, 1 × 3 min in
100% ethanol, 1 × 3 min in 95% ethanol, and air dry.
Some laboratories routinely use trypsinization or
other proteolytic treatment of formalin-fixed, paraffinembedded
tissues prior to immunohistochemistry,
e.g., 5min in 0.1% trypsin (Sigma Cat. No. T-8128 or
Dako Cat. No. $2012) in PBS at room temperature. As
stressed by Ordonez et al.
(1988), this can enhance the
staining by certain antibodies but may also result in
false negative staining with other antibodies. Thus,
control trypsinization conditions carefully and recheck
them each time a new antibody is used.
5. Treating Sections in a Microwave Oven
Cut sections onto Superfrost or similar slides. After
deparaffinization, immerse slides in citrate buffer
(2.1 g citric acid monohydrate/liter adjusted to pH 6.0
with NaOH). Microwave for two cycles of 5 min each
at a setting of 650 or 700 W. Add more buffer between
cycles so slides stay covered during the microwave
step. Cool to room temperature (cf. Cattoretti et al.
C. Histologic Staining of Sections
Stain frozen sections directly. Deparaffinize paraffin
sections (see earlier), substituting a wash with distilled
water for the air-drying step.
- Toluidine blue: Immerse sections in 1% toluidine
blue for approximately l min. Wash with distilled
water. Mount in water-soluble embedding medium,
e.g., Glycergel (Dako Cat. No. C0563).
- Hemotoxylin-eosin: Immerse sections for 10 min in
Mayer's Hemalum solution (Merck). Wash for 10 min
under running tap water, for 5 min in eosin (Merck),
and twice with distilled water and then run through
an alcohol series (e.g., 75%, 95% for 2 min each, then
100% for 5 min, then 2 × 5 min in xylol) and mount in
Eukitt (Riedel de Ha6n Cat. No. 33949) or Entellan
(Merck Cat. No. 107960).
Three methods are described in detail: immunofluorescence,
the immunoperoxidase method, and the more sensitive streptavidin-biotin method. The two
latter methods have the advantage that nuclei can be
counterstained with hemotoxylin and that only a
simple light microscope is required to visualize the
stain; however, fluorescence generally gives greater
As noted for cells, many antibodies that react well
on cryostat sections may not react on the same tissue
after it has been fixed in formaldehyde and embedded
in paraffin. In such a case it may be advantageous
to try fixing tissue, e.g., in B5, Bouin's, or Zenker's
fixative or alcohol, prior to paraffin embedding.
An interesting alternative is to use sections of
formaldehyde-fixed, paraffin-embedded material that
have been treated in a microwave oven.
For all methods mark the position of the section
after fixation; either use a diamond pencil or circle the
section with a water-repellent marker (Dako Cat. No.
S2002). Remove excess buffer after rinsing steps with
Q-tips. Use 10µl of antibody per section. Apply with
an Eppendorf pipette and use the pipette tip to spread
the antibody over the section without touching the
section. Several manufacturers (e.g., Dako) produce
excellent protocol sheets for each immunocytochemical
- Fix cryostat sections or paraffin sections deparaffinized
as described earlier for 10min in acetone at
-10°C. Air dry.
- Use steps 2-9 of the protocol given in the article
by Osborn in this volume for multitest slides. Nuclei
can be counterstained with Hoechst dye (see Osborn's
article). Positively stained cells will be green (see
Fig. 3) if an FITC-labeled second antibody is used or
red if a rhodamine-labeled second antibody is selected
(see also the double label of a cytological sample in
|FIGURE 3 Frozen section of human skin stained
keratin antibody with FITC-labeled
second antibody. Only the epidermis
|FIGURE 4 Cytological specimen from human breast
stained with a keratin antibody with
FITC-labeled second antibody
to show the tumor
cells and with a vimentin antibody and a
rhodamine-labeled second antibody to show the
other cells in the
2. Peroxidase Staining
- Fix cryostat sections for 10 min in acetone at -10°C, air dry, and wash in PBS.
- Deparaffinize paraffin sections and incubate for 30
min at room temperature in 100ml methanol containing
100µl H2O2 to block endogenous peroxidase
activity. Wash in PBS.
- Incubate for 10min at 37°C with normal rabbit
serum. Drain, but do not wash after this step.
- Incubate with primary antibody (e.g., mouse monoclonal)
for 30min at 37°C.
- Wash three times in PBS.
- Incubate with second antibody coupled to peroxidase,
e.g., rabbit antimouse for a monoclonal first
antibody (Cat. No. P0260 from Dako diluted 1:10
- Wash three times in PBS and once in Tris buffer
(6 g NaCI, 6 g Tris/liter, pH 7.4),
- Develop for 10min at room temperature using
freshly made solutions (e.g., 0.06g diaminobenzidine,
Fluka Cat. No. 32750 in 100ml Tris buffer,
0.03ml H2O2). Note: Diaminobenzidine is a carcinogen;
handle with care.
- Wash in tap water.
- Apply a light counterstain by immersing the slide
in Hemalum for 1 to 10s. Remove when staining
reaches the required intensity.
- Wash in tap water.
- Mount in Glycergel (Dako Cat. No. C0563,
- Structures that are positively stained will be dark
brown, whereas nuclei will be light blue (Figs. 5-7).
- The method can be made more sensitive by using
an additional step with a peroxidase-antiperoxidase
complex (see Sternberger, 1979).
- Blocking should be performed with a 5% solution
of normal serum from the same host species as the
labeled antibody, i.e., in this example the tissue is blocked with normal rabbit serum because the
labeled antibody is a rabbit antibody.
- Bovine serum albumin (BSA) for diluting antibodies
should be of high purity. Impure preparations
can contain bovine IgGs that can cross-react with
the labeled second antibodies.
|FIGURE 5 Paraffin section of human uterus stained with antibody
after microwave fixation with the desmin DER 11 antibody in
peroxidase technique (×160).
|FIGURE 6 Frozen section of human
after microwave treatment
with desmin antibody DEB5 and with
peroxidase-labeled second antibody.
Brown tumor cells are positive
for desmin. Nuclei are
counterstained blue (×160).
|FIGURE 1 Frozen section of human breast carcinoma
with the keratin KL1 antibody and with
antibody. Brown tumor
cells are positive for keratin. Nuclei are
counterstained blue (×150).
3. Streptavidin-Biotin Stain
Buy the reagents separately or use the Histostain kit
from Zymed Laboratories (http://www.zymed.com;
Cat. No. 95-6543 for mouse primary antibody and Cat.
No. 95-6143 for rabbit primary antibody). These kits
are based on the strong binding between streptavidin,
a 60,000-kDa protein isolated from Streptomyces avidinii
and biotin, a water-soluble vitamin (MW 244, Kd
). Instructions are given for the mouse kit.
- Fix cryostat sections for 10 min in acetone at -10°C and air dry. Sections can be treated with 0.23%
periodate for 45s. Go to step 3.
- Deparaffinize paraffin sections and air dry. Incubate
10min in PBS at room temperature and then
10min in H2O2 solution (nine parts methanol to
one part 30% H2O2 in water). Wash 3 x 2min in
- Reduce nonspecific background staining by blocking
for 10min in 10% goat serum at room temperature
(see Note 3 in Section III,D,2). Then drain but
do not wash.
- Incubate with primary antibody, e.g., mouse monoclonal
antibody, in wet chamber for 30min at
- Wash 3 × 2 min in PBS.
- Add biotinylated second antibody, e.g., goat antimouse,
for 10min at room temperature and repeat
- Add enzyme conjugate streptavidin-peroxidase
diluted 1:20 for 5min at room temperature.
This binds to the biotin residues on the second
- Develop by adding substrate-chromogen mixture
for 5min at 37°C or 15min at room temperature.
The enzyme peroxidase catalyzes the substrate
hydrogen peroxide and converts the chromogen
aminoethylcarbazole to a red, colored deposit.
- Wash 3 × 2 min in distilled water.
- Counterstain with Hemalum between 1 and 10 s.
- Wash 7min in tap water.
- Mount in glycergel.
- Structures that are positively stained will be red,
whereas nuclei will be light blue (Figs. 8 and 9).
- The optimal dilution of primary antibody has to be
determined for each antibody.
- The streptavidin-peroxidase conjugate is the same
for all species; the blocking serum and second antibody
vary according to the species in which the first
antibody is made.
|FIGURE 8 Paraffin section of human breast
after microwave treatment with
antibody in the
technique. Red tumor
cells are keratin positive.
are counterstained blue (×150).
|FIGURE 9 Frozen section of human uterus is
keratin antibody in the alkaline
technique. Only epithelial cells are positive (×150).
- The alkaline phosphatase-antialkaline phosphatase
technique is also a very sensitive method (Fig. 8).
- Both fluorescent and chromogenic signals can be
enhanced up to 1000X with Tyramide Signal Amplification
Technology (New England Nuclear). With this
technique, primarily antibodies can be diluted in some
instances more than 10,000-fold.
If possible, include (1) a positive control, i.e., a
section of a tissue known to contain the antigen; (2) a
negative control, i.e., a section known not to have the
antigen; and (3) a reagent control, i.e., a section stained
with nonimmune serum instead of the primary antibody.
Ideally, the first control should be positive and
the second and third controls negative. If high backgrounds are obtained, it may help to adjust the antibody
concentrations by increasing the length of time
for washing steps or by including 0.5M
NaCl or 5%
BSA in the antibody solutions.
Cattoretti, G., Backer, M. H. G., Key, G., Duchrow, M., Schliiter, C.,
Galle, J., and Gerdes, J. (1992). Monoclonal antibodies against
recombinant parts of the Ki-67 antigen (MIB 1 and MIB 3) detect
proliferating cells in microwave processed formalin-fixed paraffin
sections. J. Pathol
Denk, H. (1987). Immunohistochemical methods for the demonstration
of tumor markers. In
"Morphological Tumor Markers" (G.
Seifert, ed.), pp. 47-70. Springer-Verlag, Berlin.
Jenette, J. C. (1989). "Immunohistology in Diagnostic Pathology."
CRC Press, Boca Raton, FL.
Ordonez, N. G., Manning, J. T., and Brooks, T. E. (1988). Effect of
trypsinization on the immunostaining of formalin-fixed, paraffin-
embedded tissues. Am. J. Surg. Pathol
Osborn, M., and Domagala, W. (1997). Immunocytochemistry. In
"Comprehensive Cytopathology" (M. Bibbo, ed.), 2nd Ed., pp.
1033-1074. Saunders, Philadelphia.
Osborn, M., and Weber, K. (1983). Tumor diagnosis by intermediate
filament typing: A novel tool for surgical pathology. Lab. Invest
Sternberger, L. A. (1979). "Immunohistochemistry," 2nd Ed. Wiley,
Tubbs, R. R., Gephardt, G. N., and Petras, R. E. (1986). "Atlas of
Immunohistology." American Society of Clinical Pathologists