Ichthyology & Herpetology 110, No.

3, 2022, 466–476

Acid-Free Staining Procedure to Demonstrate Nerves in Whole Vertebrate

Specimens with the Differentiation of Bone and Cartilage

André L. H. Esguı́cero1,2 and Flávio A. Bockmann1,2

The value of clearing and staining whole organisms to study vertebrate anatomy is unquestionable. These methods have
been developed for over a century leading to protocols to prepare triple-stained specimens to differentiate between
bones, cartilage, and nerves. Despite their potential to advance the field of comparative anatomy, nerve-staining
methods have been used by a small number of vertebrate systematists in part because of the inconsistently successful
preparations. Here, we report on several modifications to the current Sudan black B protocols and propose a new acid-
free protocol to differentiate among bone, cartilage, and nerves in whole small vertebrates. This method may also be
used to stain solely bone and cartilage by eliminating the nerve-staining steps. The technique herein described is
successful for preparing juveniles and adults (including miniatures).

T
HE significance of anatomical studies in vertebrates used today (see modification in Steinke and Wolff, 2001),
for current scientific knowledge is immeasurable, and it has been superseded by the procedure advanced by
having its solid foundations in antiquity, in the Schultze (1897), which employs potassium hydroxide and
theories and discoveries made by Greek philosophers glycerin as clearing agents. The so-called Schultze method
(Panegyres and Panegyres, 2016). For instance, the historical has been typically used in anatomical and systematic studies
value of fish morphology extends over half a millennium, of fishes, being progressively modified through time (e.g.,
having provided the basis for its higher-level classifications Hollister, 1934; Dwight and Gore, 1936; Wassersug, 1976;
and for formulating core hypotheses about anatomical Newman et al., 1983; Mohsen et al., 2013), especially by the
evolution (e.g., Hilton et al., 2015). Vertebrate anatomy, addition of the bone-staining alizarin dye by Lundvall
especially of fishes, also has played a major role in the (1905)—whose use was greatly improved and popularized
amalgamation of cladistics as a standard methodology for by Dawson (1926)—and the cartilage-staining alcian blue dye
elucidating the phylogenetic relationships among organisms by Simons and Van Horn (1971). Further, the traditional
by providing its material framework (e.g., Bockmann et al., alkaline maceration has been modified to use enzyme
2013). Despite its current disagreement with some molecular digestion following Taylor (1967) and the subsequent
studies, vertebrate anatomy has been gaining more strength combined use of bone and cartilage staining by Dingerkus
in the last two decades and helped our understanding of and Uhler (1977) and Taylor and Van Dyke (1985). These
several fields of comparative biology (including phenotypic were great improvements that allowed researchers to obtain
adaptation, evolution, ecology, and conservation biology), much more detailed anatomical preparations that could be
showing that it is a science at full throttle (e.g., Hilton et al., stored for many years. Recent relevant improvements to the
2015). clearing methodologies applied to vertebrates were the
Despite the numerous technological advances driving our development of an acid-free protocol for fish larvae (Walker
understanding of the morphology of vertebrates in the 21st and Kimmel, 2007) and a technique for tendon staining
century (Metscher, 2009; Hilton et al., 2015; Keklikoglou et (Torres and Ramos, 2016).
al., 2019), the methodologies for clearing and staining whole In vertebrates, modern analyses of the peripheral nervous
specimens continue to play a central role in systematic system are markedly scarce when compared with the studies
investigations. Such techniques are still incredibly valuable on other anatomical complexes. For example, among the
due to the possibility of examining and manipulating various morphological synapomorphies that define the main groups
anatomical complexes in a three-dimensional perspective, as of Teleostei, only 1% belong to the nervous system, including
well as the fact that such preparations are not technologically both peripheral and central nervous systems (Datovo and
complex or costly; further, these preparations are relatively Vari, 2014). The main reason for this lack of comparative
easy to preserve. Although the impact of these techniques on studies on the anatomy of the peripheral nervous system
the field of vertebrate morphology has not yet been properly largely concerns the available techniques, which are usually
documented (Hilton et al., 2015), the protocol developed and extremely laborious (Freihofer, 1966; Filipski and Wilson,
published by Taylor and Van Dyke (1985), which is the most 1984). Pioneering studies of the nervous system of fishes
widely used for vertebrates, has accumulated 2,590 citations (e.g., Baudelot, 1883; Herrick, 1899, 1901; Allis, 1903; Ray,
in the Google Scholar database (31 July 2022). 1950) were performed by simple dissection of specimens or
The first clearing techniques were developed by Spalteholz by staining serial transverse sections through the whole fish
(1911); this technique was basically a modification of body. Both methodologies are laborious and time-consum-
histological procedures at the time, using hydrogen peroxide ing, and they require a high degree of technical skill,
with a bleaching agent (Dwight and Gore, 1936). This is little although both are necessary depending on the objectives to
1
Laboratório de Ictiologia de Ribeirão Preto (LIRP), FFCLRP, Universidade de São Paulo, Av. dos Bandeirantes 3900, 14040-901 Ribeirão Preto,
SP, Brazil.
2
Programa de Pós-Graduação em Biologia Comparada, FFCLRP, Universidade de São Paulo, Av. dos Bandeirantes 3900, 14040-901 Ribeirão
Preto, SP, Brazil; E-mail: (ALHE) andre.esguicero@gmail.com. Send correspondence to ALHE.
Submitted: 7 October 2020. Accepted: 17 January 2022. Associate Editor: W. L. Smith.
Ó 2022 by the American Society of Ichthyologists and Herpetologists DOI: 10.1643/b2020138 Published online: 26 August 2022
Esguı́cero and Bockmann—Staining nerves in whole vertebrate specimens
be achieved. The challenge of staining peripheral nerves in
small cleared whole vertebrates was addressed by Wharton
(1937) and Filipski and Wilson (1984). Beginning in the
middle of the 20th century, some anatomists began using the
Sihler’s technique which employs Ehrlich’s hematoxylin to
stain the peripheral nervous system in cleared whole mount
vertebrates (e.g., Wharton, 1937; Williams, 1943; Freihofer,
1963, 1966, 1972, 1978; Fraser and Freihofer, 1971).
Although the Sihler technique allows an exceptional visual-
ization of three-dimensional architecture of all nerves, it is
currently used by few anatomists (e.g., Mu and Sanders,
2010; Kemp, 2017) due to several limitations (Mu and
Sanders, 2010). Some drawbacks are the time the process
takes, which may take several weeks or months, the loss of
important anatomical landmarks since the decalcification of
the bone tissue prevents any bony staining, and its results are
highly dependent on the preservation technique, with
samples preserved in alcohol not being suitable for the
procedure (Freihofer, 1966).
Filipski and Wilson (1984) presented a new clearing and
staining technique using Sudan black B, which stains the
myelin sheath, as an alternative methodology to the Sihler
technique. In addition to being significantly less time-
consuming, this technique does not have a demineralization
step, therefore it preserves bones and other landmark
structures. Further, it is effective in material fixed and
preserved in either alcohol or formaldehyde. With bone
structures preserved, staining is now possible, as reported by
Filipski and Wilson (1985). However, the methodology of
Filipski and Wilson (1984, 1985) presented two problems
that were recognized by the authors themselves: the failure to
obtain good preparations with small and young specimens
and the overstaining of some fish specimens. In the Filipski
and Wilson (1984, 1985) protocol, the nervous tissue is
stained in a saturated solution of Sudan black B, followed by
destaining of tissues other than the nervous one. This
methodology was named regressive by Nishikawa (1987)
who, in turn, proposed a progressive methodology, in which
specimens are gradually stained in a 5% saturated solution of
Sudan black B in 70% ethanol, without the need for a
subsequent destaining procedure. Despite being successful in
preparing amphibian larvae, Nishikawa (1987) warned that
the long permanence of the specimens in the staining
solution as required by his technique (7 to 10 days) could
also stain other fat-containing structures and bones. Song
and Parenti (1995) altered this method in various aspects:
added cartilage staining, adjusted procedures to increase stain
retention time, detailed the concentration and immersion
time in the Sudan black B solution, and provided destaining
procedures.
The recent developments of triple differential staining
techniques for bone, cartilage, and nerves, with clearing by
means of enzymatic solutions, signaled a new universe of
anatomical investigation to be explored. However, it is rather
remarkable that such a potential has not yet been realized,
with low adherence to these methodologies among verte-
brate systematists. Indeed, in the last ten years (2012 to
2021), only 24 published works used these methodologies for
investigation of the peripheral nervous system in vertebrates
(Asaoka et al., 2012, 2014; Licht and Bartsch, 2012; Song and
Song, 2012; Boyle et al., 2013; Schomann et al., 2013;
Williams et al., 2013; Close and Cundall, 2014; Di Pietro et
al., 2014; Quinzio and Fabrezi, 2014, 2019; Hirota et al.,
467
2015; Ishida et al., 2015; Sumi et al., 2015; Sato et al., 2017,
2018, 2019, 2021a, 2021b; Schnell and Johnson, 2017;
Quinzio, 2020; Quipildor et al., 2020; Tsessarsky, 2020;
Nakae and Hasegawa, 2022). Among these, methodologies
for triple differential staining were employed in six articles
only (Song and Song, 2012; Boyle et al., 2013; Williams et al.,
2013; Di Pietro et al., 2014; Schnell and Johnson, 2017;
Quipildor et al., 2020). The long-standing tradition of
osteological studies in ichthyology, besides the relative
facility of accessing the great range of information contained
in skeleton, certainly represent a strong factor that leads most
vertebrate systematists to choose this anatomical complex
over the less accessible anatomical traits (e.g., the peripheral
nervous system). The historical paucity of studies dealing
with the peripheral nervous system anatomy resulted in a
poor comparative database, when compared to the highly
explored world of osteology, which can be another discour-
aging factor.
During the development of a research project addressing
the anatomy of the peripheral nervous system of Teleostei
representatives, we had limited success applying the existing
techniques (Filipski and Wilson, 1984, 1985; Nishikawa,
1987; Song and Parenti, 1995). The success rate in obtaining
good preparations was low in small specimens (around 50
mm SL or less), and particularly low in representatives of
Otomorpha, when compared with the results observed in
representatives of Euteleostei. We frequently experienced the
overstaining of bones and fat-containing structures while
simultaneously staining nervous tissues poorly, or the
absence of nerve staining. Thus, we performed several
modifications in the Sudan black B staining protocols already
proposed, aiming for better results. Herein, we propose an
alternative and detailed acid-free protocol for bone, cartilage,
and nerve differentiation in whole cleared vertebrate speci-
mens.
MATERIALS AND METHODS
The acid-free protocol for bone, cartilage, and nerve
differentiation in whole cleared specimens was developed
through the initial course of a project about the anatomy of
the peripheral nervous system of the Characiformes, with the
preparation of specimens of various groups of the Actinop-
terygii, in addition to some representatives of other verte-
brate groups: Caudata, Anura, and Squamata.
Specimens prepared with the methodology herein pro-
posed are part of the fish collection of the Coleção
Herpetológica de Ribeirão Preto (CHRP) and Laboratório de
Ictiologia de Ribeirão Preto (LIRP); all material was fixed in
10% formaldehyde (not always buffered) at the time of its
capture, and preserved in 70% ethanol. The longest time
interval between specimen fixation and its preparation with
the present technique was 47 years (Anchoa marinii LIRP
4365, fixed in 1974 and prepared in 2021); the standard
lengths (SL) ranged from specimens with 14.3 mm SL to
250.8 mm SL (Potamoglanis hasemani LIRP 7397 and
Rhamphichthys hahni LIRP 9422, respectively); and we also
prepared juvenile representatives of Myleus setiger (LIRP
11266), Polypterus senegalus (LIRP 15711), Prochilodus argen-
teus (LIRP 16924), Salminus hilarii (LIRP 5911), Sorubim lima
(LIRP 14527), Syacium papillosum (LIRP 16677), and tadpoles
of Bokermannohyla hylax (CHRP 2234 and 2235). One
468
miniaturized 14.3 mm SL specimen of Potamoglanis hasemani
(LIRP 7397) was also prepared.
Prepared material.—The complete listing of prepared material
is available as supplemental material (see Data Accessibility).
Solutions.—Below are detailed the necessary solutions as well
as the sequence of procedures to obtain successful cleared
and triple-stained preparations (for bone, cartilage, and
nerves) of whole vertebrates, as proposed here.
Buffered 10% formaldehyde: 1 g of calcium carbonate
dissolved in 100 ml of 10 parts of formaldehyde in 90
parts of distilled water.
Buffered 5% formaldehyde: 1 g of calcium carbonate
dissolved in 100 ml of 5 parts of formaldehyde in 95
parts of distilled water.
Ethanol (EtOH) solutions with concentrations of 25, 50,
and 75%: dilute the desired portion of ethanol in
distilled water.
EtOH-alizarin: 0.05 g of alizarin red S dissolved in 100 ml
of 75% ethanol.
Acid-free alcian blue solution: 0.02 g of alcian blue powder
dissolved in 100 ml of a solution of 5 g of magnesium
chloride dissolved in 75% ethanol.
0.1% or 0.05% trypsin solution: 0.1 g or 0.05 g of trypsin
powder dissolved in 100 ml of 1% aqueous borax
solution (i.e., 1 g of borax powder dissolved in 100 ml
of distilled water).
0.05% Sudan black B solution: 0.05 g of Sudan black B
powder dissolved in 100 ml of 75% ethanol.
Glycerin solutions with concentrations of 25, 50, and 75%:
dilute the desired portion of glycerin in distilled water.
Procedure.—Step 1. Fixation: The proper fixation of the
specimens has direct influence in the results of the clearing
and staining techniques, as detailed by Taylor and Van Dyke
(1985). The recommended fixative is a solution of buffered
10% formaldehyde (detailed in Taylor, 1977). Specimens
should remain in this solution until the tissues are fully
penetrated by it, for three or more days, depending on the
specimen size.
Step 2. Bleaching: When specimens are heavily pigmented,
bleaching is recommended. This procedure is performed with
the aid of a UV sunlamp of 300 W. The specimens are kept in
a solution of buffered 5% formaldehyde (to avoid decay) in a
bottle directly under the UV light, until it becomes
completely pale; this takes from one to three weeks,
depending on the density of pigmentation. Since the UV
sunlamp generates a lot of heat, the specimens should be
kept in a sealed glass jar close to a fan, to avoid overheating
and drying by evaporation. Due to the time-consuming
nature of this step, this procedure was only performed in
specimens with high concentrations of chromatophores.
Step 3. Cartilage stain: This step is performed in the acid-
free alcian blue solution. For the stain to penetrate the skin
more efficiently, a previous dehydration of the specimens is
necessary. This may be done by the transference through
increasing ethanol concentration solutions of 25% and 50%
(leave one hour, or longer, in each solution), and storage in
75% ethanol for at least 12 h. As the specimens are
dehydrated, they can be transferred to the acid-free alcian
blue solution. The specimens are kept in this solution until
the cartilaginous elements are stained blue, which takes
Ichthyology & Herpetology 110, No. 3, 2022
about 8 h at a temperature of 278C. After the cartilaginous
elements are stained, the specimens are rinsed in a 75%
ethanol solution in distilled water, and kept at least one hour
in this solution, for the removal of excess acid-free alcian
blue solution. The same acid-free alcian blue solution can be
used several times; solutions stored at room temperature for
two months resulted in well-stained specimens.
Step 4. Bone stain: This procedure is performed in the
EtOH-alizarin solution (detailed in Springer and Johnson,
2000). The specimens are kept in this solution until all the
bones become adequately stained, which usually takes from
about one hour, for a specimen of about 20 mm SL, to at least
6 hours, for specimens of about 150 mm SL, at a temperature
of 278C. After the bone elements are stained, which can be
checked by the visualization of easily accessible ossified parts,
such as the fins rays or branchial arches, the specimens are
rinsed in a 75% ethanol solution in distilled water, and kept
at least one hour in this solution, for the removal of excess
EtOH-alizarin solution. As the maceration, the next step, is
performed in aqueous environment, the specimens need to
be rehydrated. For this, the specimens are transferred
through decreasing ethanol solutions concentrations of
75%, 50%, and 25% (leave one hour, or longer, in each
solution).
Step 5. Preparation for maceration: Before the maceration
process, the removal of all fixative and other remaining
chemicals from the tissues of the specimens is highly
recommended because they may impair the enzymatic
action. This can be done by several changes of distilled water
or by washing the specimens in running tap water overnight,
as proposed by Song and Parenti (1995).
Step 6. Clearing: The enzymatic maceration process
(detailed by Taylor, 1967) is best carried out in a 0.1%
trypsin solution at 26–288C. An alternative to trypsin
solution that can be used is a 0.1% pancreatin enzyme
solution, but the results may not be as good due to impurities
and reduced specificity (Taylor, 1967). Further, a pancreatin
enzyme solution may require longer maceration times; this
increases the risk of bacterial and fungal contamination. This
trypsin solution concentration works fine in specimens larger
than 30 mm SL, but in smaller specimens, a lower
concentration of 0.05% is preferred, resulting in a slower
and more uniform maceration. The enzymatic action takes
from about ten hours (observed in a 26.9 mm SL specimen)
to weeks in larger specimens. To prevent the contamination
of the enzymatic solution by bacteria and fungi, the solution
should be changed every two days or more frequently if
needed. Alternatively, if temperature control is possible, the
maceration process can be carried out in a translucent
container directed under UV light, which will help to prevent
bacterial and fungal contamination of the enzymatic solu-
tion while also bleaching the specimens, in a procedure that
will not discolor previously stained bones and cartilages. The
clearing process is carried out until the specimen’s body
become translucent, i.e., when the entire vertebral column is
visible under transmitted light. The stop point of the
maceration process is a critical step. Undermacerated speci-
mens will take on too much stain in the body, preventing a
proper differentiation of the peripheral nervous system, and
overmacerated specimens will suffer damage to the myelin
sheath, with poor or even no staining of the peripheral
nervous system. An indicator of stopping maceration is when
all the bones and cartilages become noticeable under
Esguı́cero and Bockmann—Staining nerves in whole vertebrate specimens
transmitted light. If the deeper bone elements are still poorly
stained, it is possible to improve their staining at any time by
dissolving 0.01 g of alizarin red S in the trypsin solution.
When the bone elements are satisfactorily stained, the
trypsin solution should be changed. One technique to
increase the efficiency of the maceration process is to apply
an enzyme injection in deeper tissues and keep the enzyme
solution containing the specimen inside a refrigerator for a
few days, so that the solution can perfuse homogeneously
through it before starting its active digestion, when the
solution is exposed to room temperature (de Pinna, 1993).
Step 7. Preparation for nerve stain: Prior to the nerve stain,
the cleared specimens should be dehydrated by transferring
them into increasingly concentrated ethanol solutions such
as 25% and 50% for two hours, or longer, in each solution,
and keeping them in a 75% ethanol solution for at least 12 h.
Step 8. Nerve stain: The specimens are kept in the Sudan
black B solution until the nerves are pigmented with dark
blue/black, which takes from one to two days. It is
recommended to place individuals on a grid, or another
device that prevents the specimens from remaining in direct
contact with the bottom, as the decantation of Sudan black B
particles may overstain some regions of the specimens. The
Sudan black B solution can be reused multiple times, but
solutions stored for more than a month did not result in well-
stained specimens in our experience. For this reason, it is a
good practice to use fresh solutions whenever possible.
Step 9. Differentiation: Once the Sudan black B staining
process is completed, the specimens are gently rinsed in 75%
ethanol to remove excess staining solution and immediately
transferred and kept in 50% ethanol, in which removal of the
staining solution will occur slowly. The specimens must
remain in this solution until the peripheral nerves can be
recognized and before the small nerves are destained. As the
excess staining solution is removed in 50% ethanol,
peripheral nervous system differentiation continues with
25% ethanol, where destaining of surrounding tissues will
occur slowly. After differentiation of the peripheral nervous
system is finished, the specimens are transferred to distilled
water, remaining for at least one hour.
Step 10. Storage in glycerin: The final step is to transfer the
specimens through increasingly concentrated glycerin solu-
tions of 25%, 50%, and 75% (keep the specimens for at least
two hours in each solution). As reported by Filipski and
Wilson (1985), the excess Sudan black B continues to be
removed in glycerin solution so, following their recommen-
dation, the specimens should remain in the 75% glycerin
solution until the stain no longer continues to color the
solution, which must be replaced, and then proceed to
storage in 100% glycerin.
All ethanol and glycerin solutions can be reused, avoiding
only the reuse of solutions in which the presence of stain is
noticed. Glycerin solutions with concentrations under 75%
are easier to contaminate with fungi and bacteria, requiring
greater attention before being reused. It is important to
emphasize that, for every solution used during the proce-
dures, the amount to be used must be enough to fully cover
the specimens, with the volume of at least ten times that of
the specimens, as recommended by Taylor (1967). Another
guideline is that whenever applying the triple staining
protocol to several specimens at the same time, it is prudent
to choose individuals of similar body structure and volume
469
which will facilitate the procedure, because the time spent in
each step will tend to be similar in this case.
Finally, if it is necessary, the removal of remaining tissue
pigments can be taken in a 10% hydrogen peroxide and 5%
potassium hydroxide solution in distilled water, for the
removal of both guanine and chromatophores, or in a 5%
potassium hydroxide solution in distilled water for guanine
removal only. It is extremely important to highlight that
both hydrogen peroxide and potassium hydroxide can
damage the myelin sheath, so this procedure must be carried
out under strict surveillance, and it must be stopped as soon
as possible, or when the smallest stained nerve branches
become pale. This bleaching procedure tends to fill body
cavities with air bubbles, which can be removed manually or
with the aid of a vacuum pump.
RESULTS
The protocol here described results in well-cleared and triple-
counterstained specimens for bone, cartilage, and nerves, with
easy recognition of the latter, even its smallest branches (Figs.
1–3). It is important to mention that if the purpose is only to
clear and stain a specimen for its bones and cartilages but not
for its nervous tissue, the corresponding steps (steps 7 to 9)
can be suppressed without prejudice to the preparation. In
fact, the double-counterstained preparations that result from
the technique herein described are even more successful than
those that employ well-established methods (e.g., Dingerkus
and Uhler, 1977; Taylor and Van Dyke, 1985), insofar as they
also allow a better visualization of the ligaments, which
remain slightly opaque, and of the laterosensory canals and
pores, whose outlines are well demarcated.
Below, we present summary protocols for cleared and
triple-stained specimens and for cleared and double-stained
specimens (with only bone and cartilage differentiation).
Summary protocol for cleared and triple-stained
specimens
1. Fix specimens in buffered 10% formaldehyde.
2. Bleach specimens with ultraviolet radiation, if neces-
sary.
3. Dehydrate specimens through increasingly concentrat-
ed ethanol solutions of 25% and 50%, and then keep
them in 75% ethanol for at least 12 h.
4. Stain cartilages in acid-free alcian blue solution.
5. Remove excess acid-free alcian blue solution by rinsing
in 75% ethanol while keeping specimens in this
solution for at least one hour.
6. Stain bone elements in EtOH-alizarin solution.
7. Remove excess EtOH-alizarin solution by rinsing
specimens in 75% ethanol and keep them in this
solution for at least one hour.
8. Rehydrate specimens through decreasingly concentrat-
ed ethanol solutions of 75% 50%, and 25%, leaving
one hour, or longer, in each solution, and keep them in
distilled water for at least 12 h.
9. Remove all fixatives and other chemicals from speci-
mens by several changes of distilled water or washing
them in running tap water overnight.
10. Proceed to enzymatic maceration process in a 0.1%
trypsin solution.
11. Dehydrate specimens through increasingly concen-
trated ethanol solutions, such as 25% and 50%,
470 Ichthyology & Herpetology 110, No. 3, 2022

Fig. 1. Head regions, anterior to the left, of cleared and triple-counterstained fish specimens for bone, cartilage, and nerves using the technique
herein described. (A) Stenogobius genivittatus, LIRP 16424, 53.7 mm SL, left lateral view. (B) Anchoa marinii, LIRP 4365, 1, 87.4 mm SL, left lateral
view. (C) Salminus hilarii, LIRP 5911, 48.1 mm SL, left lateral view. (D) Hoplerythrinus unitaeniatus, LIRP 3945, 59.2 mm SL, left lateral view. (E)
Brachyhypopomus pinnicaudatus, LIRP 6055, 120.5 mm SL, left lateral view. (F) Trichomycterus paolence, LIRP 8274, 70.6 mm SL, dorsal view. Black
bars ¼ 2 mm. Red arrows indicate nerve branches.
Esguı́cero and Bockmann—Staining nerves in whole vertebrate specimens 471

Fig. 2. Specimens of Tetrapoda cleared and triple-counterstained for bone, cartilage, and nerves using the technique herein described.
Hemidactylus mabouia, CHRP 2238, 67.8 mm SVL: (A) dorsal view of the head, anterior up; (B) ventral view of the head, lower jaw removed, anterior
up; (C) dorsal view of the lower jaw, anterior up; (D) dorsal view of the right anterior limb, anterior down. Bokermannohyla hylax, CHRP 2236, 38.4
mm SVL: (E) dorsal view of the head, anterior up; (F) ventral view of the head, lower jaw removed, anterior up; (G) dorsal view of the lower jaw,
anterior up; (H) dorsal view of the left anterior limb, anterior down. Eurycea bislineata, CHRP 2239, 34.6 mm SVL: (I) dorsal view of the head,
anterior up; (J) ventral view of the head, lower jaw removed, anterior up; (K) dorsal view of the lower jaw, anterior up; (L) dorsal view of the left
anterior limb, anterior down. Black bars ¼ 2 mm. Red arrows indicate nerve branches.
472 Ichthyology & Herpetology 110, No. 3, 2022

Fig. 3. Cleared and triple-counterstained juvenile (A–E) and miniature (F) specimens for bone, cartilage, and nerves using the technique herein
described. (A) Bokermannohyla hylax, CHRP 2235, 34.3 mm TL, dorsal view, anterior to left. (B) Bokermannohyla hylax, CHRP 2234, 25.1 mm TL,
dorsal view, anterior to left. (C) Syacium papillosum, LIRP 16677, 26.9 mm SL, ocular side view, anterior to left. (D) Prochilodus argenteus, LIRP
16924, 40.5 mm SL, left lateral view of the head, anterior to left. (E) Polypterus senegalus, LIRP 15711, 57.4 mm SL, left lateral view of the head,
anterior to left. (F) Potamoglanis hasemani, LIRP 7397, 14.3 mm SL, dorsal view of the head, anterior to left. Black bars ¼ 2 mm. Red arrows indicate
nerve branches.
Esguı́cero and Bockmann—Staining nerves in whole vertebrate specimens
leaving them for one hour, or longer, in each solution,
and keeping specimens in 75% ethanol for at least 12
h.
12. Stain nerves in Sudan black B solution.
13. Remove excess staining solution by rinsing specimens
in 75% ethanol; transfer and maintain them in 50%
ethanol until nerve differentiation is complete; trans-
fer specimens to 25% ethanol and then to distilled
water.
14. Transfer specimens to increasingly concentrated glyc-
erin solutions of 25%, 50%, and 75%, and store them
permanently in 100% glycerin.
Summary protocol for cleared and double-stained
specimens
1. Fix specimens in buffered 10% formaldehyde.
2. Bleach specimens with ultraviolet radiation or in a 10%
hydrogen peroxide and 5% potassium hydroxide
solution in distilled water, only if necessary.
3. Dehydrate specimens through increasingly concentrat-
ed ethanol solutions of 25% and 50%, and keep them
in 75% ethanol for at least 12 h.
4. Stain cartilages in acid-free alcian blue solution.
5. Remove excess acid-free alcian blue solution by rinsing
specimens in 75% ethanol, and keep them in this
solution for at least one hour.
6. Stain bone elements in EtOH-alizarin solution.
7. Remove excess EtOH-alizarin solution by rinsing
specimens in 75% ethanol, and keep them in this
solution for at least one hour.
8. Rehydrate specimens through decreasingly concentrat-
ed ethanol solutions of 75% 50%, and 25%, leaving
them for one hour, or longer, in each solution, and
keep specimens in distilled water for at least 12 h.
9. Remove all fixatives and other chemicals from speci-
mens by several changes of distilled water or washing
them in running tap water overnight.
10. Proceed to enzymatic maceration process in 0.1%
trypsin solution.
11. Remove trypsin solution by rinsing specimens in
distilled water and transfer them to increasingly
concentrated glycerin solutions of 25%, 50%, and
75%, and store them permanently in 100% glycerin.
DISCUSSION
The successful preparation of triple-stained cleared speci-
mens depends on various factors, its prior curation condition
being one of the most significant. As reported by Taylor
(1967), specimens initially fixed in alcohol, or poorly fixed in
formaldehyde, may suffer a considerable disassociation of
tissues through the procedures. To prevent these problems,
we noted that is a good practice to do a refixation of the
specimens, prior to the clearing and staining procedures, in
buffered 10% formaldehyde, for at least one week. This
treatment gives good results, even for older specimens.
Another critical step to get better results from the nerve
staining procedure is bleaching. The use of chemical
bleaching agents, such as hydrogen peroxide, is widespread
in protocols of pigment removal from specimen bodies (e.g.,
Filipski and Wilson, 1984; Taylor and Van Dyke, 1985; Song
and Parenti, 1995). However, as reported by Freihofer et al.
(1977) and Nishikawa (1987), such a treatment negatively
473
influences Sudan black B nerve staining, as it likely destroys
the myelin sheath. This situation was also observed by us,
with hydrogen peroxide bleach rendering most specimens
poorly or non-stained. To solve this problem, we decided to
carry out the bleaching process only with ultraviolet
radiation emitted by a UV lamp. This alternative bleaching
technique was proposed by Hollister (1932, 1934) and used
by Freihofer et al. (1977) in the Sihler technique for nerve
staining, and as reported by these authors, we noted no
damage to the specimen’s tissues, as we observed when
chemical bleaching is performed. The preparations were
much superior when the bleaching process was performed
with ultraviolet radiation only, providing the effective
elimination of all dark chromatophores and guanine pig-
mentation, and clearing muscular and adipose tissues. So, the
ultraviolet radiation is highly recommended as bleaching
procedure, even though it is a slower process when compared
with chemical ones.
The traditional 30% saturated borax solution suggested by
Taylor (1967) as the solution for optimal maceration is here
replaced by the 1% borax solution, which produces the same
pH for enzyme action as reported by Taylor (1967). This
change was made with a view to facilitating the preparation
of small amounts of solutions, also resulting in reagent
savings by not requiring the prior preparation of saturated
solutions, in addition to reducing the risk of intoxication by
handling substantially smaller amounts of the chemical.
In the protocols we propose here, there is also the removal
of the potassium hydroxide from the clearing and bone stain
processes. As in the case of the use of chemical bleaching
agents, the use of potassium hydroxide for guanine removal,
clearing tissues, and as a component of the alizarin stain
solution is widely disseminated in protocols (e.g., Hollister,
1934; Freihofer, 1966; Taylor, 1967; Filipski and Wilson, 1984;
Taylor and Van Dyke, 1985; Song and Parenti, 1995).
However, as reported by Filipski and Wilson (1984) and
Springer and Johnson (2000), the use of potassium hydroxide
damages some tissues of fishes, including myelinated nerves.
The clearing and pigment removal by potassium hydroxide
becomes unnecessary with the bleaching with ultraviolet
radiation, and the bone staining can be successfully performed
in an EtOH-alizarin solution (Springer and Johnson, 2000).
The acid-free cartilage staining with alcian blue is a great
advance, by avoiding the damage to the myelin sheath by
the acetic acid action. The acid-free cartilage staining was first
proposed by Walker and Kimmel (2007) for zebra fish larvae,
and it is adjusted for larger vertebrate specimens in the
present work, allowing a better preservation of bone
elements in whole cleared specimens, even the smaller, more
fragile or poorly preserved individuals.
When testing the specimen maceration with both trypsin
and pancreatin solutions, we obtained much superior
results (faster and more crystalline preparations) with the
former. Unsatisfactory results with pancreatin solutions are
probably due to enzyme impurities that, as reported by
Taylor (1967), can decompose tissues other than the
muscular one. Another problem that we faced was bacteria
and fungus growth in trypsin solution during the macera-
tion process, which is intensified in the pancreatin solution
because, in this case, the digestion process is much slower.
In these situations, the myelin sheath was damaged,
becoming poorly stained by Sudan black B. To solve this
problem, we can change the trypsin solution frequently, or
474
add a diluted antimicrobial, such as gentamicin (40 mg
dissolved in 100 ml of trypsin solution). The use of thymol
in the trypsin solution was not tested because, according to
Song and Parenti (1995), it can negatively influence the
nerve stain by Sudan black B.
The protocols herein proposed have the potential to allow
the easy access to anatomical information of bone, cartilage,
and nervous system complexes in whole cleared vertebrate
specimens (Figs. 1, 2), adults and juveniles (Fig. 3), and even
in miniaturized forms, such as Potamoglanis hasemani (e.g.,
Henschel et al., 2018; Fig. 3F). We hope that the improved
and simplified protocol presented here for clearing and triple-
staining of vertebrate specimens, and that it yields very
successful preparations, will prompt further comparative
studies involving the vertebrate peripheral nervous system
in the near future.
DATA ACCESSIBILITY
Supplemental material is available at https://www.
ichthyologyandherpetology.org/b2020138. Unless an alter-
native copyright or statement noting that a figure is
reprinted from a previous source is noted in a figure caption,
the published images and illustrations in this article are
licensed by the American Society of Ichthyologists and
Herpetologists for use if the use includes a citation to the
original source (American Society of Ichthyologists and
Herpetologists, the DOI of the Ichthyology & Herpetology
article, and any individual image credits listed in the figure
caption) in accordance with the Creative Commons Attribu-
tion CC BY License.
ACKNOWLEDGMENTS
We thank the Programa de Pós-Graduação em Biologia
Comparada of the Faculdade de Filosofia, Ciência e Letras
de Ribeirão Preto/University of São Paulo, Brazil. Tiana
Kohsldorf and Vinicius Anelli provided us with curatorial
support at the Coleção Herpetológica de Ribeirão Preto,
CHRP (¼ Ribeirão Preto Herpetological Collection), Depart-
ment of Biology, FFCLRP/USP, Ribeirão Preto, SP, Brazil.
Photographs were taken with the aid of equipment belong-
ing to the Centro para Documentação da Biodiversidade (¼
Center for Biodiversity Documentation), Department of
Biology, FFCLRP/USP, Ribeirão Preto, SP, Brazil. This research
was funded by the Conselho Nacional de Desenvolvimento
Cientı́fico e Tecnológico–CNPq (National Council for Scien-
tific and Technological Development), Brazilian Govern-
ment, and Fundação de Amparo à Pesquisa do Estado de
São Paulo–FAPESP (¼ São Paulo Research Foundation),
Government of the State of São Paulo, Brazil (PROTAX–
CNPq #440621/2015–1 and FAPESP, #2016/50375–9). ALHE
was financed by the Coordenação de Aperfeiçoamento de
Pessoal de Nı́vel Superior, Brasil (CAPES)–Finance Code 001.
FAB was supported by Conselho Nacional de Desenvolvi-
mento Cientı́fico e Tecnológico (CNPq), Brazilian Federal
Government (#312687/2018–4).
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