General Instructions

For Immunohistochemical Staining

November 2009

For In Vitro Diagnostic Use.

Intended use and interpreted by a qualified pathologist to aid in the diagnosis
of pathophysiological processes which may or may not be associ-
For In Vitro diagnostic use.
ated with a particular antigen. (1-4)
These instructions apply to Dako immunohistochemistry reagents.
They may or may not apply to the product(s) contained in this Materials required, but not supplied
shipment.
Staining reagents, such as those provided in the Dako EnVision,
Dako antibodies are intended for laboratory use to qualitatively EnVision+, EnVision G|2, EnVision G|2 Doublestain,
identify by light microscopy antigens on or in cells from either EnVision FLEX, Dako REAL (Peroxidase and Alkaline
tissue or cell preparation samples. Positive and negative results Phosphatase detection systems), Dako LSAB+, Dako LSAB2
aid in the classification of normal and abnormal cells and tissues and Artisan Staining Systems, as well as Dako Antibody diluents,
and serve as an adjunct to conventional histopathology. The clini- and negative control reagents. Certain antibodies may require a
cal interpretation of any positive staining or its absence should more sensitive detection system such as EnVision FLEX+,
be complemented by morphological and histological studies with ADVANCE, CSA, or CSA II. Check with your distributor for
proper controls. Evaluations should be made within the context of product availability.
the patients clinical history and other diagnostic tests by a quali-
Other materials required, but not supplied include equipment,
fied pathologist. Contact Dako Technical Support to report any
chemicals and ancillary items. Equipment includes light micro-
unusual staining.
scope, staining jars or a Dako automated immunohistochemical
staining system, timer (capable of 3-40 minute intervals) and
Principles of the procedure wash bottles. Chemicals include ammonium hydroxide 15 mol/L
Dako antibodies may be used as the primary antibody with a vari- diluted to 0.037 mol/L, counterstain such as hematoxylin, distilled
ety of immunohistochemical (IHC) techniques including labelled water, ethanol (absolute and 95%), and xylene or xylene substi-
streptavidin-biotin (LSAB+ and Dako LSAB2), labelled poly- tutes. Ancillary items may include absorbent wipes; control tissue
mer and enhanced polymer systems EnVision, EnVision+, (positive and negative); coverslips; aqueous mounting medium
EnVision FLEX and FLEX+, EnVision G|2, EnVision G|2 or nonaqueous permanent mounting medium; and charged, poly-
Doublestain, ADVANCE, Dako REAL and Catalyzed Signal L-lysine coated or silanized slides. Other ancillary components,
Amplification (CSA) for the demonstration of antigens in tissue/ including control slides, may be necessary. Dako products include
cell samples. In general, IHC staining techniques allow for the target retrieval (demasking) solutions; antibody diluents; block-
demasking and visualization of antigens first by pretreatment with ing reagents; counterstains; proteolytic enzymes; chromogenic
proteolytic enzymes or heat retrieval (if required), followed by the substrates for Peroxidase and Alkaline Phosphatase; wash buffer
sequential application of a specific antibody to the antigen (pri- solutions; PAP Pen; aqueous mounting media; Silanized Slides;
mary antibody), a secondary antibody to the primary antibody (link and Control Slides. Refer to the Dako Web site, Dako catalog or
antibody or labelled polymer), an enzyme complex, and a chro- contact Dako for the most current products.
mogenic substrate with interposed washing steps. The enzymatic
activation of the chromogen results in a visible reaction product
at the antigen site. The specimen may then be counterstained
and coverslipped. Results are viewed using a light microscope,

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Storage Specimen preparation
Antibodies for IHC from Dako should be stored according to the Prior to IHC staining, tissues must be fixed and processed.
conditions detailed on their specification sheet. If using a neat Fixation prevents autolysis and necrosis of excised tissues,
(concentrated), unconjugated antibody, it may be aliquoted into preserves antigenicity, enhances the refractive index of tissue
convenient volumes and frozen at -20 C. If using an antibody that constituents and increases the resistance of cellular elements to
was frozen, avoid repeated freezing and thawing. Frozen antibod- tissue processing. Tissue processing includes dehydration, clear-
ies may be stored in small aliquots until periodic assay verifica- ing of dehydrating agents, infiltration of embedding media, embed-
tion by the user detects unacceptable changes in reactivity. (See ding and sectioning of tissues. The most common fixatives for IHC
Quality Control, Assay Verification Section.) Fresh dilutions of the tissue preparations are discussed below.
antibody should be made prior to use. Unused portions of antibody These are guidelines only. Optimal procedures must be deter-
preparations should be discarded according to local Health and mined and verified by the user. For specific information regarding
Safety regulations. Stability of diluted antibodies must be validated tissue fixation and processing, see references 1 and 6. Consult
by the user. Each antibody is suitable for use until the expiration local Health and Safety regulations.
date printed on the product label, when stored at 2-8 C.
There are no obvious visual signs to indicate deterioration of IHC Paraffin-embedded tissue
reagents. Therefore, positive and negative controls should be run General comments
simultaneously with patient specimens. If unexpected staining is
observed which cannot be explained by variations in laboratory Although 10% (v/v) neutral phosphate-buffered formalin (commonly
procedures and a problem with the antibody is suspected, contact referred to in the EU as 4% w/v buffered formalin) is the most
Dako Technical Support. common fixative, Dako Detection Systems (as listed in Materials
Required, Not Supplied) have been successfully used with tissues
Do not use products after the expiration date. If reagents are processed in a variety of fixatives. Consequently, the choice of
stored under conditions other than those specified in the package fixative is dependent on the limitations of the primary antibody and
insert, they must be verified by the user. (5) the users institutional or laboratory constraints.
The presence of turbidity and/or a precipitate in the reagent may Survival of tissue antigens for immunological staining may depend
indicate deterioration of the reagent through loss of antibody titer on the type and concentration of fixative, on fixation time, and
or due to bacterial growth. Positive and negative tissue controls on the size of the tissue specimen to be fixed. (7) It is important
should always be run simultaneously with patient specimens in to maintain optimal, standardized fixation conditions whenever
order to ascertain any loss in titer or in sensitivity. If unexpected possible in order to obtain reproducible staining. Where possible,
staining is observed which cannot be explained by variations in use of thinner specimens coupled with shorter fixation times is
laboratory procedures, and a problem with the antibody is sus- recommended. Prolonged exposure to fixatives may result in the
pected, contact Dako Technical Support. masking, impairment or destruction of antigens, which contribute
to reduced immunostaining. Zenkers fluid, B-5 and Bouins have
often been recommended as alternative fixatives for the preserva-
tion of tissue antigens sensitive to routine formalin fixation (10%
neutral buffered formalin). (8, 9) See references 1 and 6, the

4 5
primary antibody package insert(s) and the protocol(s) supplied Tissue fixation in ethanol
with the fixing reagent(s) for additional information regarding tissue Ethanol is not widely employed as a fixative for routine histologi-
fixation. cal techniques due to its poor penetrating ability. However, small
Tissue fixation in formaldehyde-based solution pieces of tissue show good cytological preservation. Fix tissue
(Neutral buffered formalin and Bouins) blocks (5.0 x 5.0 x 2.0 mm) in absolute alcohol for 48 hours at
room temperature (20-25 C) followed by two 1-hour baths in fresh
Most formaldehyde-based fixatives contain 10% formalin, a neutral
xylene and two consecutive 1-hour baths in liquid paraffin. Follow
salt to maintain tonicity, and a buffered system to maintain pH.
paraffin infiltration with embedding.
These fixatives are well tolerated by tissues and exhibit good
histological penetration. However, shrinkage or distortions may Processing and paraffin-embedding
occur in poorly fixed and embedded tissue specimens. Fix small After fixation, processing may be completed using an automatic
blocks of tissue (10.0 x 10.0 x 3.0 mm) in 5-10 mL of neutral buff- tissue processor. Tissues are dehydrated using graded alcohols,
ered formalin per block for up to 24 hours. Bouins solution is an cleared with xylene or xylene substitute, and infiltrated with paraf-
alternative formaldehyde-based fixative which contains picric acid fin wax. The tissue is subsequently embedded with paraffin wax in
and is suitable for use on all tissues except kidney. Specimens molds or cassettes which facilitate tissue sectioning. To minimize
may be fixed from 1 to 12 hours depending on tissue thickness. denaturing of antigens, do not expose tissues to temperatures in
Excessively fixed tissues become brittle and the appearance and excess of 60 C during processing. Tissue blocks may be stored
quantity of lipids is adversely affected. Complete fixation with a or sectioned on completion of embedding. Properly fixed and
70% ethanol wash to precipitate soluble picrates prior to aqueous paraffin-embedded tissues will keep indefinitely if stored in a cool
washes. place.
Mercuric-chloride containing fixatives (B-5 and Zenkers) Slides with paraffin-embedded tissue sections can be kept for up
Mercuric-chloride fixatives, such as B-5 and Zenkers, frequently to 3 years if stored at 2-8 C depending on the antigen in question.
include a neutral salt to maintain tonicity and may be mixed (11)
with other fixatives. In general, mercuric-chloride fixatives are Adherence of paraffin-embedded tissue sections
poor histological penetrators and are not well tolerated by tissue to microscope slides
specimens. Consequently, small tissue blocks (7.0 x 7.0 x 2.5 mm)
and short fixation periods (1 to 6 hours for B-5, and 2 to 15 hours Collect sectioned tissues from paraffin-embedded blocks on clean
for Zenkers) are recommended. After fixation, the tissue block(s) glass slides. Dehydrate in an oven for one hour at 60 C or less.
should be rinsed well with water and placed in 70% ethanol for wet For increased adhesion of tissue sections during IHC staining
storage or until tissue processing can be completed. Conclude fix- procedures, use of FLEX IHC Slides (code K8020), charged, poly-
ation with tissue processing and paraffin-embedding (see Process- L-lysine coated, or Silanized Slides (code S3003) is suggested.
ing and Paraffin-Embedding Section). Prior to immunostaining, When using charged, poly-L-lysine coated or silanized slides
clear tissue sections of mercury deposits using an iodine/ethanol/ specifically omit any adhesives in the mounting water bath, such
sodium thiosulfate solution. (10) Exercise the necessary precau- as gelatin, glue and/or commercially produced protein-containing
tions when handling reagents containing mercury compounds. products. Coated slides are strongly recommended for staining
procedures requiring proteolytic digestion or heat-induced epitope
retrieval (target retrieval).
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Deparaffinization and rehydration S3020), Pepsin (code S3002), or Proteolytic Enzyme, RTU (code
Prior to staining, tissue slides must be deparaffinized to remove S3007) can be used. Use the timing recommended in the specified
embedding media and rehydrated. Avoid incomplete removal of package insert of the enzyme. CAUTION: Overdigestion may
paraffin. Residual embedding media will result in increased non- result in nonspecific staining and/or unacceptable morphology.
specific or reduced staining. Rinse thoroughly with distilled water and continue with the staining
procedure of the detection system instructions.
1. Place slides in a xylene bath and incubate for 5 (1) minutes.
Change baths and repeat once. Heat-induced epitope retrieval (HIER/target retrieval) prior to IHC
2. Tap off excess liquid and place slides in absolute ethanol for staining procedures results in increased staining intensity with
3 (1) minutes. Change baths and repeat once. many primary antibodies. For some antibodies, this procedure is
3. Tap off excess liquid and place slides in 95% ethanol for 3 (1) required. Refer to the antibody specification sheet for the recom-
minutes. Change baths and repeat once. mended retrieval method. Target retrieval involves immersion of
4. Tap off excess liquid and place slides in distilled or deionized tissue sections in a pre-heated buffer solution and maintaining
water for a minimum of 30 seconds. Unless proteolytic digestion heat, either in a PT Link, water bath, steamer (95-99 C), pres-
or target retrieval is required, commence staining procedure. sure cooker (121 C), or other temperature-controlled laboratory
equipment. For optimal results on PT Link, use Target Retrieval
Xylene and alcohol solutions should be changed weekly, or after Solution from EnVision FLEX and FLEX+ kits (codes K8000,
a maximum of 200 slides. Toluene or xylene substitutes such as K8002, K8010, and K8012) or optional EnVision FLEX Target
Histoclear may be used in place of xylene; incubation times may Retrieval Solutions (codes K8004 and K8005). Other Dako target
need to be increased and the solution may need to be changed retrieval solutions include Target Retrieval Solution, pH 9 (10x),
more frequently. (3-in-1)a (code S2375), Target Retrieval Solution (code S1699 and
If necessary, rehydrated tissues may be kept in buffer solution at S1700), and Target Retrieval Solution, Citrate pH 6 (code S2369
2-8 C for up to 18 hours prior to use. Allow tissues to come to and S2031). Refer to individual instructions for use.
room temperature (20-25 C) before staining. If PT Link is used together with either Target Retrieval Solution
Proteolytic digestion and target retrieval from EnVision FLEX and FLEX+ kits or K8004, K8005, S2375,
S1699, deparaffinization, rehydration and target retrieval can be
Formaldehyde is known to induce conformational changes in carried out in one step. The PT Link run should be followed by a
the antigen molecules by forming intermolecular cross-linkages. Quick Dip procedure, using Wash Buffer from EnVision FLEX
Excessive formalin fixation can mask antigenic sites and dimin- and FLEX+ kits, Optional EnVision FLEX Wash Buffer (20x)
ish specific staining. However, these sites may be revealed with (code K8007), or Dako Wash Buffer 10x (code S3006).
proteolytic digestion or target retrieval of tissue slides prior to
immunostaining. To determine if either of these pretreatments of Using the Dako EnVision+ System, the pressure cooker method,
tissues is warranted, see the specification sheet provided with or other temperature-controlled laboratory equipment, gives
each primary antibody.
Pretreatment of tissue with proteolytic enzymes may be performed a
In the US, the Dako Target Retrieval Solution, S1699, (10x) and EnVision
prior to staining on deparaffinized and rehydrated tissue sections. FLEX Target Retrieval Solution Low pH, K8005 are not for sale as a 3-in-
Proteolytic enzymes, such as Dako Proteinase K, RTU (code 1 buffer for use in the 3-in-1 procedure. Licensing under U.S. patent No.
6,649,368 B1 may be required.

8 9
stronger staining than the water bath method. Refer to instructions Other specimens
provided with Target Retrieval Solution or reference 12.
Dako Detection Systems may also be used for staining antigens
If the water bath method is used for retrieval, some antigens may in bone sections, bone marrow, blood smears, cytospins and
require an additional pretreatment with proteolytic enzymes prior to imprints. Smears may be air-dried for 2-24 hours and processed
heating. Tissue sections can be digested with Dako Proteinase K for immediate staining or wrapped in aluminum foil and stored at
(code S3004) diluted 1:500 in a Tris-HCl buffer, pH 7.2-7.6 for 10 -20 C or lower for up to six months.
minutes at room temperature (20-25 C).
Air-dried or thawed smears may be fixed for 90 seconds in
At certain higher elevations (above 1372 m (4500 feet)), boiling acetone-methanol (1:1). Fixation in acetone or acetone-methanol-
of the target retrieval solution may occur prior to achieving the formalin (10:10:1) is also acceptable.
desired optimal temperature. In such situations, a recommended
Osseous tissues must be decalcified prior to sectioning and
alternative procedure is to heat the slides at the maximum achiev-
processing to facilitate tissue cutting and prevent damage to
able temperature and to extend the incubation time of the slides
microtome blades. (1) Decalcification may affect IHC staining.
in the target retrieval solution until the desired staining intensity is
achieved. (11) An additional possible solution is to use a closed For professional users in the United States, Clinical Laboratory
pressure system such as a pressure cooker to achieve 121 C. Improvement Amendments of 1988 (42 CFR 493.1259(b)) requires
However, each laboratory must determine the best method and that The laboratory must retain stained slides at least 10 years
target retrieval time for their particular circumstances. from the date of examination and retain specimen blocks at least
two years from the date of examination.
Frozen tissue Consult the Dako Education Guide: Immunohistochemical
Frozen sections should be cut from snap-frozen tissue blocks Staining Methods, Fourth Edition (6) or references 1 and 2 for
(approximately 1.0 x 1.0 x 0.5 cm) and air-dried for 2-24 hours. further details on specimen preparation.
Dried sections can be fixed in room temperature (20-25 C) ace-
tone for 10 minutes or in buffered formyl-acetone for 30 seconds. Precautions
Allow sections to air-dry until completely dehydrated. Proceed 1. For professional users.
with immunostaining or wrap slides in aluminum foil and store at
-20 C or lower, for up to six months. Equilibrate wrapped, frozen 2. Products may contain sodium azide (NaN3), a chemical highly
sections to room temperature prior to use. Post-fix sections in cold toxic in pure form. At product concentrations, though not classi-
acetone (2-8 C) for 10 minutes. Place slides in TBS bath. Gently fied as hazardous, NaN3 may react with lead and copper
change TBS bath several times to remove residual acetone. plumbing to form highly explosive build-ups of metal azides.
Upon disposal, flush with large volumes of water to prevent
If sections are too thick (greater than 4-6 m), incorrectly fixed or metal azide build-up in plumbing.
unevenly dried, artifacts may result and interfere with interpreta-
tion of staining. This includes rupturing of cell membranes and 3. Biological specimens, before and after fixation, and all materials
chromatolysis. Nuclei may appear swollen and uniformly blue exposed to them, should be handled as if capable of trans-
when counterstained with hematoxylin. mitting infection and disposed of with proper precautions. (14)
Never pipette reagents by mouth and avoid contacting the skin
and mucous membranes with both reagents and specimens.

10 11
 If reagents come into contact with sensitive areas, wash with Positive control tissue
copious amounts of water. Wear Personal Protective Equipment
Controls should be fresh autopsy/biopsy/surgical specimens fixed,
when handling any human biological material and while
processed and embedded in the same manner as the patient
performing the staining procedure.
sample(s). Positive control tissues are indicative of correctly pre-
4. Safety Data Sheets available for professional users on request. pared tissues and proper staining techniques. One positive control
5. Incubation times or temperatures other than those specified tissue for each set of test conditions should be included in each
may give erroneous results. Any such changes must be staining run.
validated by the user. Tissues used for positive control testing should give weak positive
6. Unused solution should be disposed of according to local, staining in order to detect subtle changes in the primary antibody
State and Federal regulations. sensitivity. Commercially available tissue slides or specimens
processed differently from the patient sample(s) validate reagent
Staining procedure performance only and do not verify tissue preparation. Refer to
the product specific package insert, Performance Characteristics
For state-of-the-art staining performance, use FLEX Ready-to- Section for normal tissue specimens that may be used for a
Use antibodies. Optimized with EnVision FLEX and FLEX+ positive control tissue.
visualization systems, these antibodies deliver desired end results
when used on PT Link and Autostainer Link or Dako Autostainer Known positive control tissues should only be utilized for moni-
instruments. toring the correct performance of processed tissues and test
reagents, NOT as an aid in formulating a specific diagnosis of
When using concentrated (neat) antibodies, refer to specific pack- patient samples. If the positive control tissues fail to demonstrate
age inserts for primary antibody dilutions and to specific Detection positive staining, results with the test specimen should be consid-
System Instructions for recommended procedures. ered invalid.
When using N-series ready-to-use antibodies, refer to the prod-
uct specific package insert and the Staining Procedure section Negative control tissue
of EnVision+, EnVision G|2 Doublestain, LSAB2 and Use a normal tissue known to be negative for the antigen being
LSAB+ Detection System Instructions. tested (refer to the product specific package insert, Performance
Characteristics Section) that is fixed, processed and embedded
Quality control in a manner identical to the patient sample(s) with each staining
Differences in tissue processing and technical procedures in the run to verify the specificity of the primary antibody and to provide
users laboratory may produce significant variability in results; an indication of background staining. The variety of different cell
regular controls need to be performed according to local guide- types present in most tissue sections offers internal negative
lines for accreditation in addition to the following procedures. control sites (this should be verified by the user).
Professional users in the United States should consult the quality If specific staining occurs in the negative control tissue, patient
control guidelines of the College of American Pathologists (CAP) specimens results should be considered invalid.
Accreditation Program for Immunohistochemistry, NCCLS Quality
Assurance for Immunocytochemistry, Approved Guideline, (15)
and reference 7 for additional information.
12 13
Negative control reagent Table 1. Examples of negative control reagents
for concentrated antibodies
Use a negative control reagent with each specimen to evaluate
nonspecific or undesired staining and allow better interpretation Primary antibody type Suggested negative control reagent
of specific staining at the antigen site. Ideally, a negative control
Monoclonal mouse FLEX Universal Negative Control for Mouse
reagent contains an antibody which exhibits no specific reactiv- Ready-to-Use antibody Primary Antibodies, code IR750 (for
ity with human tissues (non-human reactive) in the same matrix/ Autostainer Link instruments) and IS750
solution as the diluted primary antibody. The non-human reactive (for Dako Autostainer instruments)
antibody should be the same isotype and animal species as the
Polyclonal or monoclonal Universal Negative Control for Rabbit
primary antibody, diluted to the same immunoglobulin or protein rabbit FLEX Ready-to-Use Primary Antibodies, code IR600 (for
concentration as the diluted primary antibody. Normal/nonimmune antibody Autostainer Link instruments) and IS600
serum from the same species as the primary antibody, at a protein (for Dako Autostainer instruments)
concentration equivalent to the diluted primary antibody in the
Monoclonal mouse antibody, Non-human reactive monoclonal antibody
same matrix/solution may be suitable for use depending on the produced in ascites produced in ascites.This negative control
type of primary antibody used. Refer to the package insert of each antibody should be of the same isotype
primary antibody and to Table 1 for specific recommendations. as the primary antibody. Alternatively,
Diluent alone may be used as a less desirable alternative to the normal/nonimmune mouse serum may be
previously described negative control reagents. The incubation used, Dako code X0910
period for the negative control reagent should be equivalent to Monoclonal mouse antibody, Non-human reactive monoclonal mouse
that of the primary antibody. produced in tissue culture antibody produced in tissue culture.This
negative control antibody should be of the
same isotype as the primary antibody.
Dako codes X0931 (IgG1), X0943
(IgG2a), X0944 (IgG2b), X0942 (IgM).
Alternatively, fetal calf serum may be
used.*
Polyclonal rabbit antibody, Rabbit Immunoglobulin fraction (Normal),
immunoglobulin fraction Dako code X0903
Solid-phase absorbed Rabbit Immunoglobulin Fraction
polyclonal rabbit antibody, (Solid-Phase Absorbed),
immunoglobulin fraction Dako code X0936
Polyclonal rabbit antibody, Normal/nonimmune rabbit serum,
whole serum whole serum, Dako code X0902
* Fetal calf serum is suitable for use if it is retained in the primary antibody
after processing.

14 15
When panels of several antibodies are used on serial tissue Table 2. The purpose of daily quality control
sections, the negatively staining areas of one slide may serve Tissue: fixed Specific antibody & Negative reagent control*
as a negative/nonspecific binding background control for other & processed detection system or buffer plus same
antibodies if their dilutions are similar and they are from similar similar to detection system as used
animal sources. patient sample with specific antibody

To differentiate endogenous enzyme activity or nonspecific bind- Positive Control: Controls all steps Detection of non-specific
ing of enzymes from specific immunoreactivity, additional patient Tissue or cells of the analysis. background staining.
known to contain Validates reagent
tissues may be stained exclusively with substrate-chromogen or target antigen to and immunostaining
enzyme complexes (avidin-biotin, streptavidin, labelled polymer) be detected (could procedures.
and substrate-chromogen, respectively. For specific procedures, located in patient
contact Dako Technical Support. tissue). Tissue which
exhibits weakly positive
staining is most
sensitive to antibody
or detection system
degradation.
Negative Control: Detection of Detection of non-specific
Tissue or cells unintended antibody background staining.
expected to be cross-reactivity to
negative (could be cells/cellular
located in patient components.
tissue or positive
control tissue).
Patient Tissue Detection of Detection of non-specific
specific staining. background staining.
* Same species and isotype as the specific antibody, but not directed against
the same target antigen. To detect non-specific antibody binding, e.g.,
binding of Fc portion of antibody by the tissue.

Assay verification
Prior to initial use of an antibody or immunostaining system in
a diagnostic procedure, the user should verify the antibodys
specificity by testing it on a series of in-house tissues with known
IHC performance characteristics representing known positive and
negative tissues. Refer to the quality control procedures previ-
ously outlined in this section of the General Instructions and, for
US professional users, to the quality control requirements of the
16 17
CAP Accreditation Program for Immunohistochemistry and NCCLS Negative control tissue
Quality Assurance for Immunocytochemistry, Approved Guideline. The negative control tissue should be examined after the positive
(15) These quality control procedures should be repeated for control tissue to verify the specificity of the labelling of the target
each new antibody lot, or whenever there is a change in assay antigen by the primary antibody. The absence of specific staining
parameters. Tissues listed in the product specific package insert, in the negative control tissue confirms the lack of antibody cross-
Performance Characteristics Section are suitable for assay veri- reactivity to cells/cellular components. If specific staining, other
fication. than that described above, occurs in the negative control tissue,
results with the patient specimen should be considered invalid. In
Troubleshooting negative tissues, the tissues should have a blue-purple appear-
Refer to the Troubleshooting section in the Dako Education Guide: ance when using hematoxylin.
Immunohistochemical Staining Methods, Fourth Edition (6) for Nonspecific staining, if present, will be of a diffuse appearance.
remedial action, or contact Dako Technical Support or the website Sporadic staining of connective tissue may also be observed in
at www.dako.com to report unusual staining. sections from excessively formalin-fixed tissues. Use intact cells
for interpretation of staining results. Necrotic or degenerated cells
Staining interpretation can exhibit nonspecific staining. (7)
Positive control tissue False-positive results may be seen due to non-immunological
The positive control tissue should be examined first to ascertain binding of proteins or substrate reaction products. They may also
that all reagents are functioning properly. Positive reactivity is indi- be caused by endogenous enzymes, such as myeloperoxidase,
cated by the presence of a red (3-amino-9-ethylcarbazole, AEC), leucocyte alkaline phosphatase and hemoglobin pseudoperoxi-
bright pink (New Fuchsin or Fast Red) or brown (3,3-diaminoben- dase, primarily in frozen tissues and depending on the type of
zidine tetrahydrochloride, DAB) reaction product at the site of the enzyme label used to visualize the reaction. (17)
target antigen. See the Staining Interpretation and Performance Patient tissue
Characteristics sections of the product specific package insert
for specific staining patterns. If the positive control tissues fail to Patient specimens should be examined last. Positive staining
demonstrate the expected staining pattern, all results with the test intensity should be assessed within the context of any nonspe-
specimen should be considered invalid. cific background staining with the negative control reagent. As
with any IHC test, a negative result means that the antigen was
NOTE: The color of the reaction product may be different if not detected, not that the antigen was absent in the cells/tissue
substrate chromogens other than those stated are used. Refer to assayed. If necessary, use a panel of antibodies to identify false-
the substrate package insert for expected color reactions. Further, negative reactions.
metachromasia may be observed in variations of the method of
staining. (16) Refer to the product specific package insert for information
regarding primary antibody immunoreactivity.
Depending on the incubation length and potency of the hematoxy-
lin used, counterstaining will result in a blue coloration of the cell
nuclei. Excessive or incomplete counterstaining may compromise
proper interpretation of results.

18 19
General limitations 6. Tissues from persons infected with hepatitis B virus and con-
taining hepatitis B surface antigen (HBsAg) may exhibit non-
1. IHC is a multi-step diagnostic process that requires specialized
specific staining with horseradish peroxidase. (18)
training in the selection, fixation and processing of tissue;
selection of reagents; preparation of the IHC slide; and inter- 7. Reagents may demonstrate unexpected reactions in previously
pretation of the staining results. untested tissues. The possibility of unexpected reactions even
in tested tissue groups cannot be completely eliminated due to
2. Tissue staining is dependent on the handling and processing
biological variability of antigen expression in neoplasms or
of the tissue prior to staining. Improper fixation, freezing,
other pathological tissues. (19) Contact Dako Technical Sup-
thawing, washing, drying, heating, sectioning or contamina-
port with any documented unexpected reaction.
tion with other tissues or fluids may produce artifacts, antibody
trapping, or false-negative results. Inconsistent results may be 8. Normal/nonimmune sera from the same animal species as
due to variations in fixation and embedding methods, or to the secondary antisera used in blocking steps may cause false-
inherent irregularities within the tissue. negative or false-positive results due to autoantibodies or
natural antibodies.
3. Excessive or incomplete counterstaining may compromise
proper interpretation of results. 9. False-positive results may be seen due to non-immunological
binding of proteins or substrate reaction products. They
4. The clinical interpretation of any staining, or its absence must
may also be caused by endogenous biotin or enzymes, such
be complemented by morphological studies and proper con-
as myeloperoxidase, leucocyte alkaline phosphatase and
trols as well as other diagnostic tests. It is the responsibility of
hemoglobin pseudoperoxidase, primarily in frozen tissues and
a qualified pathologist who is familiar with the antibodies,
depending on the type of immunostain used. (17)
reagents and methods used to interpret the stained prepara-
tion. Staining must be performed in a certified licensed labora- 10. Heat induced epitope retrieval (target retrieval) may result in
tory under the supervision of a pathologist who is responsible HIER lipofuscin artifacts. Target retrieval may result in demask-
for reviewing the stained slides and assuring the adequacy of ing of unexpected or undesired sites.
positive and negative controls. 11. False-negative staining results, with or without background,
5. Unexpected negative reactions in poorly differentiated neo- may be encountered if the concentration of primary antibody
plasms may be due to loss or marked decrease of expression is too high when used in a given staining system.
of antigen or loss or mutation(s) in the gene(s) coding for the 12. Ready-to-use primary antibodies are prediluted and optimized
antigen. Unexpected positive staining in tumors may be from for use with specific staining systems. When used in conjunc-
expression of an antigen not usually expressed in morpho- tion with other Dako or manufacturers detection systems these
logically similar normal cells, or from persistence or acquisition are no longer ready-to-use and must be re-optimized and
of an antigen in a neoplasm that develops morphologic and validated according to the clinical laboratory IHC protocol.
IHC features associated with another cell lineage (divergent
differentiation). Histopathologic classification of tumors is not 13. Unless specifically claimed in the instructions, the performance
an exact science and some literature reports of unexpected characteristics of antibodies used for IHC have not been deter-
staining may be controversial. mined for other laboratory techniques.

20 21
References 12. Shi SR, Key ME, Kalra KL. Antigen retrieval in formalin-fixed,
paraffin-embedded tissues: An enhancement method for
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