Advanced Concepts in Fixation: 1.

Effects of
Fixation on Immunohistochemistry , ,.

Reversibility of Fixation and Recovery of

Proteins, Nucleic Acids, and other Molecules
from Fixed and Processed Tissues.
2. Developmental Methods of Fixation
Isam ~ltoum:::',Jerry Fledenburgh" William E. ~ r i z z l e '
Department of Pathology, University of Alabama at Birmingham, Birmingham, AL
' Richard Allan Scientific, Kalamazoo, MI

Abstract Introduction
The generally i~nprovedunderstanding of b i o c h e ~ ~ ~ i c a l In a prior malluscript in this issue, we discussed the basic
pathways and genetic alterations that ~tnderliemany disease mechanisms of vario~istypes of fixation and some of the
processes have led medical scientists to rely increasingly on factors that affect fixation, such as time, temperature, pH,
the identification of specific molecular markers that are im- specimen size, and tissue characteristics (1). In this article,
portant for diagnosis and lnanagernent of diseases. This mo- we focus on some of the more colnplex issues of fixation,
lec~tlarapproach requires optinla1 preservation of proteins, including the effects of fixation 011 i~nmunol~istoche~~~istry,
RNA, and DNA in diagnostic specimens of tissue. How- over- and underfixation, the potential reversibility of mo-
ever, the standard tissue preparation designed for optimum lecular changes induced by the fixatives that cross-link, fu-
histo~norphologicalexamination constit~~tes several steps, ture developments in fixation, and changes in fixation
none of which aims at good recovery of protein, RNA, or needed to support new methods of molecular analysis.
DNA. This article reviews the more co~nplexissues of fixa-
tion, i~lcludillgthe effects of fixation on irnm~inohistochem- Effects of Fixatives on Immunohistochemistry
istry and recovery of RNA, DNA, and proteins fro111 fixed Fixation alone does not characteristically cause a loss of
tissue. We also discuss over- and ~~nderfixation, ~nicrowave immunorecognition of antigens; instead, i~nmunorecogni-
fixation, perfusion-fixation, and the potential reversibility of tion for some antigens is lost after specific combinations of
~nolecularchanges induced by formaldellyde and other fixa- fixation, tiss~teprocessing, and paraffin embedding. Thus, a
tives. We stress the need for designing fixatives rationally fixative that works just fine in irnmunohistochemical iden-
so that their effects are known and could be controlled and tification of an antigen on frozen sections (eg, neutral buff-
reversed when needed. (Tlze J Histoteclzrlol 24:201, 2001) ered formalin (NBF) fixation and Bcl-2 imm~unostaining)
may not work following the same NBF fixation plus sub-
Key words: lnicrowave fixation, lnolecular changes, mo- sequent processing, paraffin embedding, and preparation of
lecular markers, overfixation, perf~ision-fixation,i~nderfix- paraffin sections, even when the same exact monoclonal
ation antibody is used in both staining procedures.
There is no best fixative or fixative-processing combina-
tion for immunorecognition of all antigens; rather an anti-
Address reprint requests to Isa~nEltoum, MD, Department of Pathology,
University of Alabama at Birmingham, Kracke Building-Room 627, Uni-
gen-and antibody clone-and a fixative-processing-
versity Station, Birmingham, AL 35294. Phone: (205)-934-4590; Fax: e m b e d d i n g method must be matched to optimize
(205)-934-7094 immu~lorecognition(2). When a fixative that does not pre-

The Journal of Histotechnology IVol. 24, No. 3 1 September 2001

. . serve antigen recognition is used, various approaches may
"reverse" specific molecular changes produced by fixation.
This theory of reversal of the effects of fixation is discussed
in this article, and the Elias article in this issue discusses
practical application of antigen recovery (3).
If processing-embedding is matched (ie, maintained con-
stant), the effects of fixation on paraffin embedded material
can be demonstrated; and it can be shown that the choice of
fixation may have a large effect on irnmunorecognition. Our
prior studies, which compared the effects of 7 different
fixatives under the same processing-embedding regimen,
evaluated the immunorecognition of 5 antigens in adeno-
carcinolnas of the human breast and colon under different
conditions of fixation (2). We found that either unbuffered
10% formalin or zinc unbuffered 10% formalin was the best
fixative (ie, gave the strongest immunostaining) for
p185"rbB-' and TGFcu. In contrast, for p53 and cytokeratins
(AEl/AE3), alcoholic formalin, 95% ethanol or 100%
methanol gave good and equivalent results. These gave bet-
ter results than 10% NBF, unbuffered formalin, Bouin's, or
zinc unbuffered formalin. Of the fixatives compared, in gen-
eral 10% NBF resulted in the poorest immunorecognition
(Figure I). Not only may the use of different fixatives result
in different intensities of immunostaining, but, in some
cases, different patterns of immunostaining. A specific ex-
ample is that the expression of p185"'bB-' has an increased
expression of p185""bB-' on cell membranes and solnewhat
decreased expression of p185"-bB-' in the cytoplas~nof cells
of breast cancer. As discussed, this may cause problems
with the Herceptin test (4). The etiology of this change is
unknown.
In a separate study of the CWR22 xenograft of prostate
cancer, we found that PenFix (alcoholic formalin) was a
much better fixative for dernonstrating EGF receptor and
pi85"r"B-' than was 10% NBF (5).
Effect of Fixation on Special Stains of Histochemistry
The main effect of fixatives on special stains in histo-
chemistry is the loss during fixation and processing of spe-
cific lnolecules that a r e targets of the histochemical meth-
ods. This usually is caused by the dissolution of these
molecules. Typically, some ~noleculesare soluble in aque-
ous fixatives (eg, glycogen, catecholamines), whereas oth-
ers are soluble in organic based fixatives (eg, lipids). Also,
there may be modification of staining secondary to changes
in pH induced by fixation, some fixatives chemically
modify the targets of the histoche~nicalstain or induce new
targets nonspecifically, such as the effect of glutaraldehyde
fixation on silver stains (6).
The effects of fixation with glutaraldehyde are iluportant

T I FORMALIN
0.80
ACID
FORMALIN
W 0.60
d ZINC
0 F0RMN.M
2 0.40
Em33 ALCOHOLIC
FOIWLIN
0
zz 0.20 13'1-IANOI.
4
$ 0.00
KEIWI'INS
m MELHANOL
0 p53
I SEM

TAG-72 p 185 erbB-2 TGF-ALPHA

Figure 1. The effects of different fixatives on the intensity of immunostaining of 5 antigens are demonstrated: p53, AEVAE3 cytokeratins, tumor associated
glycoprotein-72 (TAG-72). p185"""-2, and TGFa. SEM = standard error of the mean.

Advanced Concepts in Fixation: Effects on IHC, Recovery of Molecules, and Developmental Methods 1 Eltoum et al
to consider. An active chemical group, a free aldehyde is
introduced by glutaraldehyde's reactions with amine, am-
ide, sulphydryl groups, or alcoholic R groups of proteins.
This is because, unlike formaldehyde, glutaraldehyde has a
free amine group on each end of the molecule. As discussed
in the prior article, aldehyde groups are very active, and free
aldehyde groups can reduce silver salts in basic solutions
(1). Thus, when argentaffin (Fontana-Masson) or argyrophil
(Grinielius) stains are performed, silver is deposited at the
aldehyde groups added by the reactions of glutaraldehyde
with protein side chains in addition to the specific aldehyde
groups that are the targets of argentaffin methods and that
also react in argyrophil stains. This may result in an increase
in background staining in tissues fixed in glutaraldehyde
and stained with silver methods (6). PAS stains also may be
affected.
One of the best correlations of the effects of fixation on
histochemistry is tabulated in Chapter 2, Fixation, in Tlzeoiy
crild Pinctice of Histotechizologj by Sheehan and Hrapchak
(7). We have modified this table so that harmful methods of
fixation can be identified rapidly, and this table is included
in the prior article by Eltoum et a1 (1).
Over- arzd Uizderlfixation
Because of the rush to process specimens for a rapid
diagnosis by s ~ ~ r g i c apathology,
l some thick (4-5 mm)
specimens may be underfixed, especially in their center.
This may be observed in the paraffin blocks as an indented
center of the block, on paraffin sections as poor staining and
autolysis in the center of sections, and/or by "holes" in the
center of paraffin sections because the middle tissue is too
autolytic to hold together. If the underfixation is less severe,
both irn~n~~nohistochemical and histochemical staining will
be inadequate in the center of the tissue sections, but such
staining inay be improved by fixing sections before stain-
ing. This is accomplished by cutting sections, attaching the
sections to slides, removing paraffin from the sections, and
rehydrating the specimens to buffer. At this point, the speci-
mens are returned to a bath of NBF for varying periods up
to several hours. The 10% NBF is removed by washing in
tap water, and the sections are returned to buffer prior to
staining.
Overfixation and/or overprocessing may first be recog-
nized as "hard" specimens after tissue processing. Such
hardness is especially a problem in fixatives containing de-
hydrants, including ethanol, methanol, or acetone. Thus,
overfixation usually presents as a specimen that is difficult
to cut, especially a specimen that shatters on cutting. This
may be a problem, especially with animal tissues that re-
quire shorter processing protocols to reduce their dehydra-
tion. If animal tissues or small biopsies (1-2 mm) are pro-
cessed routinely at 1 hr or more per processing bath on a
typical tissue processor, the specimens may become too
hard. For animal tissues that contain no fat, setting each
processor solution at 30 min to 40 Inin should correct the
problem. See the cornpanion article in this issue by Grizzle,
Stockard, and Billings (4).
The increased hardness resulting from overdehydration
may be partially corrected by cutting into the tissue and then
soaking the tissue in water. Water may penetrate up to 0.5
mm into the block, and this re-hydration acts to "soften" the
block. Other approaches to rehydrating " h a r d tissues have
The Journal of Histotechnology I Vol. 24, No. 3 1 September 2001
been proposed, including soaking the tissue in various so-
lutions as follows:
Horrze's Softeiziizg Agent
95% ethanol
Glycerol
Acetone 40 ml
80% phenol solution 40 ml
Overfixation with formaldehyde or other cross-linking
fixatives results in tissues with poor imm~inohistochemical
and/or poor histological staining. If the tissue has been fixed
for weeks or longer, overfixation may not be totally cor-
rectable; however, overfixation may be reversed partially.
As discussed, the introductory article on fixation in this
issue, washing tissue in water may stop and partially reverse
fixation. The extent of reversing overfixation or fixation in
general depends upon the stability of cross-linking.
Advanced Theory of Fixation and Reversibility
of Cross-Linking
Advanced Corzcepts of Formaldehyde Fixation
Reactions of FormnkEEhyrle with MLicro171olccules
(Figure 2 )
At low pH, formaldehyde forms the reactive electrophilic
carbonium ion (+CH,OH) that is believed to initiate most of
the formaldehyde reactions (8,9). At high pH, 2 methylene
molecules (2CH,(OH),) may combine through Cannizza-
ro's reaction, which recently has received some interest be-
cause it explains the liberation of hydrogen from aqueous
formaldehyde under alkaline conditions (10). It is interest-
ing to note that the amino group (-NHZ), the most common
reactive nucleophile in tissue, reacts more slowly with form-
aldehyde at acid pH than basic pH because of the formation
of a charged -NH, group at low pH.
Reactions of formaldehyde with ~nacromoleculesare nu-
merous and complex (9,I 1-15). Formaldehyde reacts with
active hydrogen groups forming hydroxymethyl (-CH,OH)
adducts, which may condense with other active groups to
form methylene cross-bridges between macromolecules via
inter- and intramolecular cross-linking of macromolecules.
Formaldehyde penetrates between nucleic acids and pro-
teins and stabilizes nucleic acid-protein shells (16-19). It
modifies nucleotides by reacting with free amino groups of
nucleotides as in proteins. In naked and free DNA, the
cross-linking reactions are believed to start at adenine-
thymidine (A-T) rich regions, and cross-linking increases
with increasing temperature (16-19). Formaldehyde reacts
with C=C and -SH bonds in unsaturated lipids; however, it
doesn't interact with carbohydrates (8,9).
Revecribility of Fori~ialdehyde-Macror?zoleculesReaction
(Figure 3)
Fraenkel-Conrat and his colleagues frequently noted that
the addition and the condensation reactions of formaldehyde
with amino acids and proteins were unstable and could be
reversed easily by dilution or dialysis (1 1-15). The com-
paratively stable remaining cross-links have been attributed
to the reactions between amino-hydroxymethyl groups and
amide or guanidyl groups, forming a N-C-N bond (Model
Equation I), and with indole, phenol, or imidazole groups,
 I P-NI-I?- + HCHO e--
-+
Reversible
reactions
+

Figure 2. Reversible, acid-labile and acid resistant proteins-formaldehyde reactions

Model equatiolr I , acid-labile lir~k:

P-NH, + CI-I,O P-NH-CH,OH + P-NH, ---+
P-NH-CH,-NHP + H,O
Model eqllntio112, acid-resistallt link:
P-NH, + CH,O ! I & P-NH-CH,OH + P-R' ---b P-NH-CH, -R'-P + H,O
Model equation 3, secondary anli17eblocks o-oss-lii~ki~zg:
R2 -NH +CH,O R2N-CH,OH + P-NH, -+ R2N-CH,-NH-R-P + H,O
Moclel equatio~a4, pi-inzaly allzille a~lglizeiztsacid-labile c~.oss-li~zkiilg:
R-NH,+ CH,O RNH-CH,OH + CH,O RN-(CH,OH), + 2P-NH, -+
(P-NI-I),-NR +2H,O
Figure 3. Model equations of the interactions of formaldehyde, anlines and proteins (P-NH,). R-NH2 = an a ~ n i n oacid or a primary amine. R2-NH =
Secondary amine. R' = indole, plienol or imidazole group. R,NH = secondary amine.

forming a N-C-C bond (Model Equation 2). Indole-
formaldehyde and some methylphenol-formaldehyde cross-
links can be hydrolyzed by strong alkali (5 hr, 120°C, with
5N NaOH). However, even under these conditions, phenol-
formaldehyde links are very stable. On the other hand, his-
tidine-formaldehyde links can be hydrolyzed with acid (1 8
hr, 120°C, 6N HCI).
Tome and his colleague used carbon- 13 n~tclearmagnetic
resonance (NMR) spectroscopy to confirm what Fraenkel-
Conrat had observed. Using bovine serum albumin as a
model protein, they found lysine to be the most frequent
amino acid involved in formaldehyde-protein interactions
(20,21). Lysine-arginine, lysine-glutarnine, or lysine-
asparagine constitute the most common acid-labile cross-
links, whereas lysine-tyrosine cross-links resist acid hydro-
lysis. In the absence of lysine residues (acetylated albumin),
formaldehyde forms links with other amino acids (20,21).
Methylene cross-links between the indole ring of histadine
or between the imidazole ring of tryptophan and other
groups, possibly amide or terminal NH2 groups, are acid
labile (20,21).
~ o I e c ~ ~ l l e dehyde, cause extensive protein cross-linking (Model Equa-
Marzipulntiorz o f F o ~ i ~ c ~ l d e l z y d e - M c ~ c ~ ' o ~ iReactions
Schwendeman et al proposed a simple model for protein-
fortnaldehyde interactions (Figure 1) (22). Using amino
acid analysis, they noticed re~narkablechanges in the amino
acid composition of proteins during the storage of toxoid
vaccines; specifically, they observed a reduction of lysine,
histidine, and tyrosine residues in aggregated compared to
unaggregated toxoid. These workers established the role of
formaldehyde in this aggregation and were able to stabilize
the toxoids by blocking both the reversible and irreversible
f.'ormaldehyde reactions through succinylation of lysine resi-
dues or through reductive tnethylation of hydroxyrnethyl
adducts (22). These findings prove that one can manipulate
fortnaldehyde interactions to produce specific effects. Thus,
the reactions summarized in Figure 1 can be manip~ilatedto
block the irreversible reactions in favor of the reversible
reactions. The fixed tissue can then be manipulated to re-
lease specific macromolecules.
Secondary atnines or primary amides form a single hy-
droxymethyl residue when reacted with formaldehycle
(Model Equation 3). Therefore, they "cap" the side chain
amino group and inhibit cross-linking (13-15). It is possible
that these ~noleculescan be used to both reduce formalde-
hyde-protein cross-links and to improve the retrieval of
macroniolec~~les at the tissue level if added as a separate
step in fixation. On the other hand, primary arnines, which
form 2 hydroxymethyl residues when reacted with fornlal-
tion 4). The cross-links formed with amino compounds,
such as analine, glycine, cyclohexylamine, serine, and arn-
moniun~chloride are acid labile N-C-N bonds (Figure 1). A
few studies have shown that addition of phenol, lysine, cy-

204 Effects on IHC, Recovery of Molecules, and Developmental Methods 1 Eltoum et a1

Advanced Concepts ~n F~xat~on:
clohexylamine, or niercaptoethanol improved and acceler-
ated fixation by NBF (23,24). Therefore, to favor the reac-
tion of for~naldehydewith the exogenous cotnpound over
unwanted endogenous residues, such as tyrosine (which
forms acid-resistant cross-links), one could select amines
with higher affinity for formaldehyde, ie, with higher reac-
tion constants (Kc[), such as glycine, as well as molar con-
centrations of the amine that will not colnpromise the pro-
cess of fixation.
Revei:~iOili~y r!f' Links wit11 Mlcleic Acid
Because the reactions of formaldehyde with nucleic acids
are similar to those with proteins, changing the temperature,
pH, and constituents of fixation or extraction buffers may
assist in recovering nucleic acids. Inside the cell, RNA and
DNA are closely associated with proteins; during fixation,
formaldehyde forms RNA-protein, DNA-protein, and pro-
tein-protein cross-links (25). The reversibility of these links
has been extensively explored in gellotoxic and structural
studies, which utilize lower formaldehyde concentrations
(1% or less) than the 4% solutions used ill tissue fixation for
diagnostic purposes. Jackson has shown that after treating
nuclei with 1% forinaldehyde at 4°C at pH 7.4, for a few
minutes to 2 days, protein-DNA cross-linking can be re-
versed completely by incubating for 2 days in 0.1 % SDS/5O
mM Tris, pH 8.8, at 37"C, or by incubating for 2 hr in the
same solution at 60°C (26,27).
Jackson preferred the former treatment because the latter
procedure resulted in considerable nicking of nucleic acids.
Jackson noted that the reversal of protein-protein cross links
requires much harsher treatment to elicit enough resonance
to break the cross-links, eg, heating at 95°C and the use of
2-mercaptoethanol to prevent oxidation of proteins at this
high temperature. Jackson also noted that during the steps of
fixation, a high pH favors protein-protein over protein-DNA
cross-linking and that the addition of exogenous amines
stops the reaction between nucleic acids and proteins.
Casanova-Schmitz and Heck, itsing enzymatic digestion,
approximately doitbled the total amount of extracted DNA/
RNA, which was found entrapped as DNAJRNA-protein
cross-links within the interface between the aqueous and the
organic phases of the guanidinium-phetiol-chlorofor111 ex-
traction mixture (28). After prolonged fixation of naked
DNA (40 days, at 4"C), Chaw identified nucleoside-CH2-
nucleoside cross-links using enzyme digestion and reverse-
phase high-pressure liquid chromatography. These links
form 6% of total RNA nucleosides and 2% of total DNA
~iucleosides.Although they are unstable in acid, the links
can be hydrolyzed with heating to 60°C for 5 hr or 90°C for
1 hr, or in alkaline solution in the presence of sodium boro-
hydride at 60°C (29).
Varioi~sexplanations have been put forward to explain
the failure of RNA recovery from forrnalin fixed materials,
including degradation of RNA during processing, excessive
cross-linking with proteins or with phenols and guanidinium
thiocyanate present in the extraction fluids, and modifica-
tions that inhibit reverse transcriptase reactions and subse-
quent PCR. Successfi~lattempts at recovering RNA and
DNA fro111 tissue fixed in 10% NBF include digestion with
proteinase K to remove proteins, heating at 70°C to remove
hydroxymethyl adducts, and heating with the addition of a
chelating agent to remove cross-linking metals and DNase
The Journal of Histotechnology IVol. 24, No. 3 1 September 2001
to retllove contaminating DNA (30). Masuda et al have
reported that formaldehyde adducts inhibit cDNA synthesis
and that simple heating reverses this reaction (3 1). Although
high concentrations of formaldehyde or prolonged fixation
cause direct DNA-DNA cross-links, it is not known if these
links occur during tissue fixation. Fortunately, if they do
form, these links call be cleaved by sodiu~llborohydride or
by heating in 1 N hydrochloric acid (26,27,32,33).
The addition of exogenous amines or amino acids could
improve the fixation process via a~nine-DNAcross-links
and inhibition of protein-DNA cross-links (26,27). The
DNA-DNA cross-links formed with methylamine, dimeth-
ylamine, diethylamine, or morpholine break readily in aque-
011s media, but not in organic-aqueous media or in the pres-
ence of excess forfiialdehyde (34). As in proteins, the
N-C-N cross-links between nucleic acids and amines or
amino acids can be easily hydrolyzed.
Knowing the conditio~is-such as temperature, concen-
tration of reactants, and buffer pH-under which the re-
versible, acid-labile, and acid resistant reactions occur is
essential for devising a better strategy of retrieval of mac-
romolecules. For example, during formaldehyde-protein in-
teractions, the irreversible formaldehyde content increases
with increasing pH while the reversible remains constant
(35). The work of Fraenkel-Conrat and his colleagues forms
the bases of antigen retrieval inethods that have remarkably
changed the practice of imtnitnoliistochemistry. Factors that
influence antigen retrieval include (36-38):
@ Temperature: High temperature remains one of the
most important factors in antigen retrieval, though a
low tenlperature antigen retrieval system has recently
been shown to work for some antigens. Both the extent
a i d time of heating are important. In general, the
higlier the temperature, the better the immunostaining;
a result that can be obtained in 10 min at 120°C may
take 10 hr at 70°C.
@ pH: Different antigens give different results clepencli~ig
on pH of antigen retrieval fluids. Shi et a1 classifiecl
antigens into the following: stable type that changes
slightly with pH, variable type that gives results at
extremes of pH, and ascending type that improves with
increasing pH. They suggested that antigen retrieval at
high pH may be 1i1ost itsefi~l.
Molarity: Molarity is not irnportarit for some buffers.
ow ever, when aluminurn hydroxide is used, 4% has
been shown to work better than other concentrations.
@ Presence of metals: Though in their first work, Shi and
associates suggested that metals play a role in antigen
retrieval through re-fixation of antigenic determinants,
they later reported that metals are not a necessary com-
ponent of antigen retrieval solutions (36-38).
Reve~ribilityof' Class-Links Fornzecl D~lrir~g Fixation with
Fixatives Other- T11ar1Fouilnlclehyde
Aldehydes: The reactions of glutaraldehyde and macro-
molecules are generally accepted as it-reversible. There are
few studies about the seversibility of cross-links developed
during fixation with other aldehydes, such as acrolein or
glyoxal. A l t h o ~ ~ gwe
h don't know which of its reactio~ls
predominates during fixation, acrolein (HC=CH.CHO) re-
acts with macron~oleculesin a manner similar to formalde-
hyde, forming liyd~oxymethylreactive groups, which then
condense with other amino groups to form cross-links.
Theoretically these links will be reversible under the con-
ditions discussed above for formaldehyde (8).
NH-P '
Unlike formaldehyde, acrolein reacts readily with fatty acid
through the double bond.
HC=CH.CHO + R-COOH a R-COO.CH,CH,.CHO.
Cyclic and more co~nplexproducts may then form. These
reactions are probably not reversible.
Because of acrolein extreme reactivity with macromol-
ecules, Hayat recommeild its use in histochemistry only
when forrnaldehyde and glutaraldehyde fail to produce ad-
equate results (8). Like, glutaraldehyde, acrolein adds a car-
bony1 group to the macro~iloleculeand thereby reacts posi-
tively with Scliiff's reagent.
Glyoxal, diformyl, (CHO.CH0) is another bifiuictional
aldehyde that reacts almost identical to formaldehyde. Each
of the formyl groups can react with nucleophile.
CHO.CHOH-NH-P + H2N-P'+ CHO-NH-P + H 2 0
I as glutaraldehyde. Because of these features, their versatil-
NH-P'
The glyoxal reaction with nucleosides has been extcn-
sively studied because of potential genotoxic effects of this
co~npound.It reacts readily with adenosine, cytosine, meth-
ylcytocine, and methylguandine, forming DNA adducts in
an unstable atid reversible manner (39). Thus, glyoxal
should be handled with care.
Recently, glyoxal has received new attention as a poten-
tial formaldehyde substitute for routine fixation because it
produces few fumes, particularly if it is to be used in mi-
crowave fixation.
Carbodiimides: Carbodiimides (RN=C=NR1) are deriva-
tives of cyanamide (NH=C=NH), a known condensing
agent that may be regarded as a symmetrical anhydride of
urea (NH,-CO-NH,). Since their description in the 19th
century, these compounds received great attention in a va-
riety of fields (40). They react with Inany organic and fi~nc-
tional groups including amino, alcoholic, phenolic, phos-
phate, and sulphydryl residues. However, these reactions
can be enhanced or selectively blocked by choosing the
appropriate type of carbodii~nides,pH, temperature, solvent,
or catalyst (40). For example, a high temperature favors
reactio~iswith alcohols. At pH 7.0-7.5, the predominant
reaction occurs between carboxylic acids and amino acids.
This latter reaction is probably the most exploited reaction
particularly in peptide synthesis and i~n~nunogen prepara-
tion.
The reaction starts with the interaction of carbodiimides
with carboxyl groups forming an 0-isourea derivative that
is vulnerable to nucleophilic attack by adjacent amino
206 Advanced Concepts in Fixation: Effects on IHC, Recovery of Molecules, and Developmental Methods I Eltourn et a1
groups. This then can for111 a peptide bond, and a hydrated
form of carbodii~nideis released.
RN=C-HNR' + H,N-P'+ RNH-C-HNR' + P-C-P'
I Il II
OOC-P 0 0
If this reaction occurs during tissue fixation, as has been
widely suggested, then there is an opportunity for breaking
tlie peptide bond formed during cross-linking, which wo~tld
be susceptible to broad-spectruni or specific enzyme diges-
tion. DNA and RNA could then be released from tissue
through enzymatic digestion. Addition of an exogenous di-
amine will not only augment the cross-linking but may irn-
part some specificity to the peptide bond formed; selection
of a specific protease can break the bond without much
damage to the rest of the structure of the protein. Because
they have little effect on antigenic determinants of irnmu-
nogens, these co~npou~ids were tried as tissue fixatives first
and successfi~llyfor itnmu~iohistoche~nistry (41).
Subsequently, several workers have found that these
compounds are good for routine histology, enzyme liisto-
chemistry, and electron tnicroscopy (4 1 4 5 ) . Ya~ilamoto
and his associates found these coiilpou~ldswere better than
formaldehyde or glutaraldehyde at maintaining tissue en-
zyme activity while preserving tissue ultrastructure as well
ity and the potential of reversing their effects on tissues, we
think these cornpounds should be revisited as fixatives for
routine histology.
Diimidoester: These are water-soluble compounds that
interact very rapidly with proteins, forming amidine. The
a~nidineformed can be hydrolyzed easily with a ~ n m o ~ i i a
(46). These compounds have been reported to be good fixa-
tives for both electron tnicroscopy and immunohistochem-
istry (46).
Metallic fixatives: Salts and complexes of zinc, chro-
miurn, mercury, osmiurn, palladium, and uraniu~nhave all
been utilized as fixatives. All react with various reactive
organic resiclues and create tnultiple cross-links with the
metal in the center of tlie cross-link. The reversibility of
these reactions is not well studied. Powerfi~lgeneral chelat-
ing agents or specifically designed approaches iiiay reverse
some or all the effects of metallic fixatives 011 ~~iacromol-
ecules.
Developnlent of An Ideal Fixative
In addition to its illiportance in advancing in medical
science by allowilig recovery of intact, un~nodifiedmacro-
molecules from fixed tissues, any fixative should inhibit
autolysis of tissue, stop the growth of bacteria and infectious
agents, maintain tissue and cellular integrity, and prevent
diffusion of substances away from their natural sites. In
addition, the ideal fixative should have the following attrib-
utes (47):
0 It should be effective for a wide variety of tissues and
organs, including fatty tissue, soft lyrnphoid tissue,
 neural tissue, and small and large specimens of differ-
ent animal species.
@ It should be suitable for all procedures and tests in a
histology laboratory: routine stains, special stains, irn-
munostains and hybridization techniques.
@ It should give the best morphological res~lltsin the
hernatoxylin and eosin (H&E) stain, and the stained
sections sho~tldbe stable on storage.
0 It should be stable, with a shelf life of at least 1 yr.
@ It should be compatible with most tissue processors.
It should penetrate and fix t i s s ~ ~ine a realistic time.
It should be convenient to use and save, without being
extremely flammable.
@ It should be ~tsablefor long-term specimen storage.
@ It should pose minimal toxicological risks under rou-
tine laboratory use.
@ It must be cost-effective.
@ It should be readily disposable and recyclable.
0 It should fix tissues so that they cut easily when em-
bedded in paraffin.
OLISimproved understanding of biochemical pathways
and genetic alterations that underlie many disease processes
have led us to increasingly rely on the identification of
specific molecular markers that are important for diagnosis
and management of diseases. This ~nolecularapproach re-
quires optimal preservation in chemically unmodified forms
of proteins, RNA, and DNA. However, the standard tissue
preparation for optimuln histo~norphological examination
constitutes several steps, none of which aims at an optimal
recovery of protein, RNA, or DNA from fixed and paraffin
embedded tissues. In fact, one may argue that there will
always be a contradiction or a paradox between keeping
morphology intact and optimizing the recovery of macro-
molecules, because the latter constitutes the bricks and nor-
tar that keep the histological structures intact. Consequently,
the worse the fixative is in allowing recovery of RNA,
DNA, and proteins, the better it preserves morphological
and, especially, ultrastruct~~ral details. Glutaraldehyde is a
good example. Using unprocessed tissues, such as frozen
sections, is an example of the reverse, ie, the potential for
good recovery of rnacrornolec~tlesbut poor morphology.
Pathologists and technologists should push the idea of
rational fixative design to produce a better neth hod of fixa-
tion in the 21st century. Understanding how fixatives work
will help in selecting and designing the best way to reverse
their effects. Fortunately, most of the fixatives react with
tissue component in different ways, depending on the con-
ditions of fixation. As we have discussed, formaldehyde
reactions with tissue can be manipulated to favor reversible
reactions. Glyoxal and other aldehyde interactions with tis-
sue also may be exploited to "zip" and "unzip" bonds in a
predictable and controlled fashion; for example, cross-links
produced by carbodiimides are peptide bonds that can be
broken easily by proteases. This calls for a better under-
standing of the interactions of fixatives at both molecular
and tissue levels.
Special Methods of Fixation
Aizimal and Tissz~ePerfusion
It is freq~ientlyu s e f ~ to
~ l "fix" all the tissues of an animal
quickly and concomitantly or to fix all the components of a
The Journal of Histotechnology IVol. 24, No. 3 ISeptember 2001
whole organ. Infusing the whole animal with a fixative of
choice may do this. The blood must first be removed from
the animal's vascular system by perfusion, preferably with
4°C saline. Removal of the blood is required; otherwise the
fixative will clot the blood in the vascular system so that it
cannot be replaced by the fixative. To perfuse the saline and
s~~bsequently the fixative through the animal's vascular sys-
tem requires "force," which is obtained by raising the con-
tainers of the perfusion solutions about 36 inches above the
animal. For perf~lsion,the chest of a deeply anesthetized
animal is opened and the needle from the saline line is put
into the left ventricle of the animal's heart; then the left
atri~unof the heart or pulmonary veins are cut for an exit of
the blood-saline. When clear saline comes from the left
atrium, the fixative solution should be shifted to a needle in
the left ventricle.
For a rodent, 100 ml of fixative will perfi~seall organs;
larger animals may require 500 ml of fixative. Organs can
be removed fro111 the perfi~sedanimal under a hood to mini-
mize exposure to vapors from the fixative, and gloves
should be worn to minimize exposure of the skin to any
fixative.
To specifically fix the brain, perfitsion is facilitated by
tying off the descending aorta. To fix the lungs (to evaluate
pulmonary fibrosis, emphysema, cancer, or structural de-
fects), it is best to inflate-fix by tying off the trachea and
pumping the fixative into the trachea distal to the tie. This
method of inflate-fixation will prevent the evaluation of
pulmonary edema and may wash inflammatory cells and
other debris from the tracheobronchial system into the al-
veolar spaces; this artifact of fixation can usually be recog-
nized by ciliated col~unnarcells mixed with the inf-lamma-
tory cells. Pulmonary edema can be evaluated by taking a
section of lung before inflate-fixation and by clamping the
cut areas with surgical clips or a hemostat prior to intlate-
fixation.
Issues in Fixatiorz for Electron Microscopy
Volume Chnrzges on Fiscltiorz
Electron microscopy and cyto~norpho~netric studies are
used to determine and evaluate sizes of cells and cellular
organelles. However, the size of cells and cellular organ-
elles are sensitive to fixative osmolality, and sizes may
change on fixation because of shrinkage or swelling (48).
For example, ~nusclesof the frog may shrink LIP to 45%
upon fixation (49). In contrast, Ehrlich ascites turnor cells
shrink in all formaldehyde-glutasaldehyde combinations,
swell in 4% for~nalinor I% os~niumtetroxide, but remain
unchanged in 3% glutaraldehyde or 3% glutaraldehyde plus
1% osmium tetroxide. Neurosecretory granules change in
size based on the osmolality of the o s ~ n i u ~tetroxide
n fixa-
tive (50).
II~CI'LICL'~~
Ai.t(fc~firctsqf Str~lct~~t-e by Fixntiorl
The most prominent artifacts induced by fixation are at
the ultrastructural level. For example, membrane-vesicle fit-
sion seen in some cells examined by electron lnicroscopy
was previously thought to be related to cellular fi~nction.It
has since been demonstrated to be due to induction by glu-
taraldehyde fixation of fusion of probable extracellular
vesicles with cellular membranes (51). Similarly, what ap-
peared to be vesicles in cells of the ear in osmium tetroxide
fixed tissue has been shown to be degraded interdigitations
of cell membranes when tissues were preserved in glutar-
aldehyde (52). Glutaraldehyde has been shown to nonspe-
cifically bind horseradish peroxidase to cellular surfaces;
this may appear as a false positive reaction for antigens in
itnrni~nohistochemicalprocedures (5334). Gli~taraldehyde
fixed tissues also can give false patterns of staining in ar-
gyrophil/argentaffi11 and PAS procedures (6). Similarly, the
demonstration of enzymatic activity is quite variable, de-
pending on the fixative (55). Although the method of fixa-
tion is important to interpretation of ultrastrilctural ele-
ments, the largest problem in determining actual structures
based on electron microscopy is the interpretation of a 3
di~nensionalstructure in 2 dimensions. Multiple different
structures may give the same 2-dimel~sionalrepresentations
based on the plane of section chosen. This further compli-
cates the identification and interpretation of artifacts in-
duced by fixation.

Loss of Molec~llesor1 Fisatiorl

The smaller the molecule, the Inore likely it is that the
molecule will be lost from tissue during fixation or subse-
quent tissue processing. This is not so big a problem in
routi~iestaining, which usually is not focused on smaller
molecules. However, staining of endocrine tu~norsmay be
changed by loss of small moleciiles at both the light and
~~ltrastructural
levels. For example, adrenal medullary tissue
fixed in glutaraldehyde will lose all epinephrine as well as
metabolites of norepinephrine. Fixation in formaldehyde at
pH 7.0 results in the loss of about 60% of catecholamines
from neurosecretory granules. Similarly, up to 97% of small
neuropeptides (eg, leuteinizing hormone releasing hormone
(LHRH)) are soluble in etl~anoland may be lost in dehy-
drant fixatives, during dehydration or processing, or after
staining. In contrast, fixation with glutaraldehyde plus di-
chromate prevents the loss of epinephrine and ~i~aintains Figure 4. This shows a ductal atlenocarcinoma immunostained for DR-5
receptor of'TRAIL and counterstainetl with light hernatoxylin. Panel A ( X
catecholamines in other granules. 400) demonstrates moderate to strong cytoplas~nicstaining of DII-5. Panel
Some fixatives such as osmium tetroxide 111ay cause the B ( X 400) demonstrates an area adjacent to that shown in Panel A. This area
degradation of specific proteins, including actin filaments demonstrates severe cautery artifact, stretched out and wavy nuclecrr shapes
and keratin (56,57). Osmium tetroxide also may act to leach (arrows). Only in this area was the cytoplasmic staining of DR-5 negative.
proteins from cellular membranes (58). Similar loss of staining in areas of cautery artifact was noted in other cases
with this and other antibodies.

Trozible Shootirzg Problems with or overfixation (fracture). If there is no evidence of prob-

Fixatiorz-Tisszie Processirzg
Frequently problems with i1111ni11~0histochemica1, histo-
chemical, or ~~ltrastructural staining are attributed to prob-
lems with "fixation," but as discussed in the cornpanion
article by Grizzle et al, niay be due to multiple factors,
including con~binationsof the various processes used to
prepare tissues for staining to staining, or to tissue damage
that occurs before fixation (4). For example, surgeons fre-
quently may "cut" tissues with a range of tools from lasers
to the electric cautery. These tools may cause localized
tissue damage that may manifest itself by poor staining and,
in sonle cases, by recognizable histopathological (eg, mor-
phological) changes (Figure 4). These changes in tissue are
irreversible, and the important issue is to recognize such
areas as being damaged and not usable for evaluation of
i~nm~~nohistochemical or histochemical staining or struc-
tural analysis. Similarly, if an area of a tissue stains poorly,
the block should be examined for evidence of under- (col-
lapsed center of block or autolysis in the center of the block)
lems with the block, the staining procedures should be re-
peated, insuring that the procedure is followed carefully and
that all reagents are in date. It also will be itsefi~lto check
for similar proble~nswith other stains 011 sections from this
same block. If 110 laboratory problems are identified, it may
be necessary to evaluate whether or not the same problerns
always arise from the same source of tissue. For example,
someone at that source may be placing the fresh tissue on
dry absorbent material (dry sponge or paper), may be leav-
ing the tissue too long in saline or other solutions prior to
the tissue being fixed, or may be permitting the tissue to dry
prior to being sent to the laboratory. Problem solving for a
specific source will require a walk-through by laboratory
personnel following a specimen from step to step.
Microwave Zrradiatiorz irz Fixation
Since its first use as a method of fixation in 1970, the
microwave gradually has foiund its way into histology labo-
ratories and this development has revolutionized many as-

Advanced Concepts In Flxatlon: Effects on IHC, Recovery of Molecules, and Developmental Methods 1 Eltoum et al
pects of histotechnology (59). The microwave, an instru-
ment that produces non-ionizing radiation with a frequency
of about 2.45 GHz, generates heat through molecular oscil-
lations produced when microwave radiation passes through
such dipolar molecules as water. This heat is thought to play
a major role in microwave-induced fixation. Unlike the heat
generated by boiling o r other means that is conducted, heat
induced by the ~nicrowaveat the molecular level is less
subject to variation throughout a tissue. T h e heat generated
to produce temperatures in the range of 45-70°C is adequate
to produce optimal fixation; thus, this range is optimal for
the denaturation of proteins (55). In addition to heat, 1110-
lecular oscillations may disrupt hydrophilic and hydropho-
bic bonds and other forces that maintain macromolecular
structure and, hence, morphology. For example, Hjerpe and
his associates f o u n d that reactions between antigen-
antibody (CEA-anti-CEA)-a non-covalent bonding-
proceeded 1000 time faster during microwave irradiation,
even when they maintained a cool temperature during the
reaction (60). Hopwood suggested that fixation induced by
irradiation appeared to be accomplished by denaturation,
unraveling of si~lphydrylgroups, and subsequent disulfide
bridging (55).
A major advantage of the use of the microwave is its
ability to shorten the time required for histopathological
evaluation; after the first step of gross examination of the
tissue, use of the microwave may shorten all steps of tissue
fixation and processing as well as the final step of histo-
logical staining (61). Large specimens are often difficult to
fix in a short period of time because of the limitations of
diffusion. Prolonged fixation of the whole specimen in such
a case may result in uneven fixation, with a hard periphery
and soft, partially autolytic center. It has been suggested that
fixation using a microwave could reduce the time of speci-
men processing, ensure uniformity of fixation, preserve
~norphologyof large specimens, improve im~nunostaining,
and decrease D N A degradation (61). During gross exami-
nation, large specimens such as the brain, stomach, and
intestine could be rendered firm to improve slicing and sam-
pling without loss of tinctorial characteristics by immersion
in normal saline followed by microwave irradiation to tem-
perature of 68-74°C.
Microwave irradiation of tissue to prepare it for histo-
logical exa~ninationcan be accomplished in several differ-
ent ways, including the following (61):
@ Microwave the tissue in normal saline without using a
chemical fixative.
@ Cover the specimen with a primary fixative such as
10% N B F and irradiate it with the microwave for vari-
ous empirically determined times
@ Microwave the specimen in saline followed by chemi-
cal fixation.
@ Chemically fix and follow by irradiation of the speci-
men in saline.
@ Microwave to aid in fixing frozen sections prior to
staining.
The microwave can be used in subsequent processes like
dehydration and clearing of paraffin infiltration. After par-
affin sections are cut, the microwave also can be used to
accelerate staining processes as well, especially in silver
staining (6,62,63). Microwave technology can have an im-
portant impact on turn-around times without significantly
The Journal of Histotechnology I Vol. 24, No. 3 I September 2001
jeopardizing histomorphology. How well the microwave
alone o r in combination with chemical fixation permits
maximum recovery of DNA, RNA, and protein need to b e
studied further.
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