Received: 9 November 2018 Revised: 16 February 2019 Accepted: 10 March 2019

DOI: 10.1002/jemt.23263

RESEARCH ARTICLE

A protocol for processing the delicate larval and prepupal

salivary glands of Drosophila for scanning electron microscopy
Denisa Beňová-Liszeková | Milan Beňo | Robert Farkaš

Laboratory of Developmental Genetics,

Institute of Experimental Endocrinology, Abstract
Biomedical Research Center, Slovak Academy Although scanning electron microscopy (SEM) has been broadly used for the examination of fixed
of Sciences, Bratislava, Slovakia
whole insects or their hard exoskeleton-derived structures, including model organisms such as
Correspondence
Drosophila, the routine use of SEM to evaluate vulnerable soft internal organs and tissues was
Robert Farkaš, Laboratory of Developmental
Genetics, Institute of Experimental often hampered by their fragile nature and frequent surface contamination. Here, we describe a
Endocrinology, Biomedical Research Center, simple four-step protocol that allows for the reliable and reproducible preparation of the larval
Slovak Academy of Sciences, Dúbravská cesta and prepupal salivary glands (SGs) of Drosophila for SEM devoid of any surface contamination.
9, Bratislava 845 05, Slovakia.
Email: ueenfark@savba.sk
The steps are to: first, proteolytically digest the adhering fat body; second, use detergent washes
to remove contaminating coarse tissue fragments, including sticky remnants of the fat body; third,
Review Editor: George Perry
use nonionic emulsifying polysorbate emulsifiers to remove fine contaminants from the SGs sur-
Funding information
Agentúra na Podporu Výskumu a Vývoja, face; and fourth, use aminopolycarboxylate-based chelating agents to detach sessile hemocytes.
Grant/Award Number: APVV-16-0219; EEA Short but repeated rinses in 100 μL of a saline-based buffer between steps ensure efficient
Grants, Grant/Award Number: Norwegian FM
removal of remnants removed by each treatment. After these steps, the SGs are fixed in glutaral-
SK-0086; European Cooperation in Science
and Technology, Grant/Award Number: ENBA- dehyde, postfixed in osmium tetroxide, dehydrated, critically point-dried, mounted on aluminum
CA15216; FP7 Research infrastructures, stubs, sputter coated with gold–palladium alloy and examined in the SEM.
Grant/Award Number: INSTRUCT-
FP7-211252; North Atlantic Treaty
KEYWORDS
Organization, Grant/Award Numbers: CRG-
972173, LST.CLG-977559; Vedecká Grantová delicate tissue, dirt removal, Drosophila melanogaster, salivary glands, SEM, surface
Agentúra MŠVVaŠ SR a SAV, Grant/Award
contamination
Numbers: 2/0103/17, 2/0109/13

1 | I N T RO D UC T I O N tissues of holometabolous insects undergo serious reconstruction or

Preparing the delicate and small-dissected organs or tissues from
insects, even those of well-established model organisms, for detailed
microscopic observation raises a unique set of challenges. In contrast to
the hardened exoskeleton and exoskeleton-derived structures of adult
insects where a combination of common detergent and mild ultrasonic
cleaning can be used to remove surface dirt, the internal organs and tis-
sues require a different approach, as they are too soft and fragile to sur-
vive this treatment. This problem has not been fully solved even
though scanning electron microscopy (SEM) is the most widely used of
all electron beam technologies in biomedical research and its challenges
recognized as soon as SEM began to be used to examine the surface
structure of soft tissues (Al-Tikriti, Henry, Al-Bagdadi, Hoskins, &
Titkemeyer, 1986; Bozzola & Russell, 1999; Dickson, McKenna,
McColl, & Carr, 1989; Echlin, 2009; Kuo, 2014; Watt, 1997). This prob-
lem is important to experimentally characterize the cellular proliferation
and de novo morphogenesis of organs and tissues during different
developmental stages, and absolutely critical to understand how the
complete histolysis during the transition from larval to adult life stages
(Bayer, von Kalm, & Fristrom, 1996; Gilbert, Rybczynski, & Tobe, 1996;
Hagedorn, 1985; Ianella, Azeredo-Oliveira, & Itoyama, 2008; Jurand &
Pavan, 1975; Poulson, 1950; Riddiford, 1993; Riddiford, Hiruma, Zhou, &
Nelson, 2003; Schooley, Horodyski, & Coast, 2005; Truman & Riddiford,
2002). While the structural examination of soft tissues by SEM is often
hindered by the presence of contaminating dirt from histolyzed body
parts, this is especially the case when these tissues are dissected from
stages undergoing metamorphosis. Indeed, the majority of historical as
well as current knowledge on the morphological changes seen in meta-
morphic processes comes from histological or transmission electron
microscopical studies in which the gross morphology, internal structure,
organogenesis, or ultrastructure of particular tissues and cells has been
studied using a series of semi-thick or ultrathin sections. These focus
mostly on the inside of tissues so that there is no interference from
contaminants associated with other dissected tissues or cellular debris
from histolyzed organs (Berendes & Ashburner, 1978; Brandão Ados
et al., 2014; Farkaš, 2016; Farkaš & Šuťáková, 1998; Gautam, Verma, &

Microsc Res Tech. 2019;1–12. wileyonlinelibrary.com/journal/jemt © 2019 Wiley Periodicals, Inc. 1

2 BEŇOVÁ-LISZEKOVÁ ET AL.
Tapadia, 2015; Kuleesha, Puah, & Wasser, 2016; Lane, Carter, &
Ashburner, 1972; Lockshin & Zakeri, 2001, 2004; von Gaudecker, 1972;
von Gaudecker & Schmale, 1974). Most of the available studies made by
SEM focus on an animal's external gross morphology and only to a lesser
extent their internal organs (Corwin, Clifford, & Keirans, 1979; Harrison,
2012; Jenkins, 1991; Li, Guo, Xu, Li, & Han, 2017; Speirs, White, &
Wilson, 1986; Stueben & Linsenmair, 2009). Those focusing on internal
organs usually use tissues dissected from stages where there is no sub-
stantial tissue remodeling under way, and so there is minimal generation
of cellular debris. Moreover, if only a few representative or exemplary
images of internal organs are required, then a standard protocol may be
adequate examining a few to dozens of samples on a few stubs is often
sufficient to produce enough high-quality images without disturbing
surface contamination. This kind of “trial and error” approach is unac-
ceptable, however, for the intensive and systemic study of dynamic pro-
cesses, notably during the turbulent metamorphic period when multiple
tissues simultaneously undergo programmed histolysis. For these rea-
sons, we developed a procedure to routinely obtain high-quality tissues
for SEM that is free of surface-dirt contamination arising from adherent
foreign tissue components and cellular debris of various origins. We
found out that introducing three or four short chemical treatments into
a standard tissue-processing protocol for SEM substantially diminishes
unwanted surface contamination. Following a brief enzymatic digestion,
the dissected tissue is chemically treated with diluted detergents, emulsi-
fiers, and/or chelatonic complexons. These simple modifications allow
for the routine production of reproducible and good-quality SEM images
from soft internal organs. Here, we demonstrate the successful applica-
tion of this protocol to the salivary glands (SGs) of Drosophila, but it can
be easily modified for other tissues dissected from different develop-
mental stages, and should have versatility for application to various other
non-drosophilid and even non-dipteran insect species as well.
2 | MATERIALS AND METHODS
2.1 | Fly culture
Wild type (Oregon R) fruit flies (Drosophila melanogaster, Meigen) were

cultured in 50-mL vials or 200-mL bottles at 23 C on agar-yeast-corn-
meal-molasses medium (Ashburner & Thompson, 1978; Ransom,
1982) with the addition of methylparaben to prevent molds. The
experiments described here used the SGs of wandering last larval
instar and prepupal stages.
2.2 | Tissue dissection and treatments
Extrafine Dumont #5 tweezers were used to dissect SGs from larvae or
prepupae placed in a drop of Drosophila saline (Ephrussi & Beadle, 1936)
on a glass slide while viewed using a Wild M3Z or Leica MZ9.5 stereomi-
croscope. After the adhering fat body was removed mechanically as
much as possible using the forceps, the glands were transferred to a
fresh drop of saline. When no enzymatic or chemical treatment was
applied to the SGs, they were immediately fixed in 4% paraformalde-
hyde + 2% glutaraldehyde in 100 mM sodium cacodylate buffer (pH 7.2)
at 23 C and further processed for SEM as described below.
We tested different approaches to remove or at least minimize
surface contamination of the SGs by various types of dirt—either
material and cells circulating in the hemolymph during metamorphosis
or material generated during the dissection procedure, some of which
has a relatively strong tendency to adhere to the basement membrane
of tissues. Based on our previous successful use of glycosidic enzymes
to remove strongly adherent dirt from the integument of pharate
adults who were unable to successfully eclose from the pupal case fol-
lowing metamorphic development (Beňo, Liszeková, & Farkaš, 2007),
we assessed the effectiveness of three approaches for the removal of
strongly adhering foreign material. We first tried a different group of
enzymes, mostly proteinases, then detergents and polysorbate emulsi-
fiers that could minimize surface tension thereby releasing dirt adher-
ing through a complex of weak nonspecific organic interactions, and
finally chelating agents (aminopolycarboxylic acids) such as EDTA and
EGTA to minimize interactions mediated by divalent, usually inorganic,
cationic interactions. Since all of the enzymatic and chemical treat-
ments were done after tissue dissection and prior to aldehyde fixation,
we attempted to minimize the side effects of performing these treat-
ments on unfixed tissue by performing them as quickly as possible.
First, we evaluated enzymes that are most frequently used in the
sophisticated preparation of primary cell cultures by tissue dissocia-
tion and maceration. These were used either individually or as a mix-
ture of two or more enzymes, notably when similar digestion
conditions, close to optimal, could be used. The following enzymes
were evaluated: 0.005% trypsin (Sigma #T-4174, Steinheim,
Germany), 0.0025% chymotrypsin (Calbiochem #230832, Schwalbach,
Germany), 5 μg/mL proteinase K (Qiagen #19131, Hilden, Germany),
0.005% elastase (Sigma #E-7885, Steinheim, Germany), 0.15 mg/mL
collagenase I (Sigma #C-0130, Steinheim, Germany), 25 μg/mL
pronase (Roche Diagnostics #10165921001, Mannheim, Germany),
and 0.01 mg/mL dispase (Worthington Corp. #LS02100, Lakewood,
NJ, USA). When an enzyme was used individually, we used a buffer
giving optimal activity; mostly this was provided or recommended by
the manufacturer. When all seven enzymes were used together, this
was not possible so enzymatic treatment was performed in Drosophila
saline. In contrast to the methods used for tissue dissociation, we
always incubated tissues with the enzyme(s) for 1–2 min at ambient
temperature (23 C). Immediately after digestion, the incubation solu-
tion was removed and glands were washed at least five times for
15–30 s each with fresh Drosophila saline Ringer.
Early in the course of this study, we found out, not unexpectedly,
that the majority of enzyme buffers are incompatible with the presence
of many detergents, especially sarkosyl and deoxycholate. Conse-
quently, detergent treatments were applied after the washes following
enzymatic digestion, as follows: 0.1% saponin (Acro Organics/Janssen
Chimica), 0.001% SDS (Sigma), 0.1% Triton X-100 (Serva GmbH.),
0.05% sarkosyl (Serva GmbH.), 0.001% sodium deoxycholate (E. Merck)
for 2 min at 23 C. We found that saponin had no significant effect on
releasing dirt, while SDS was too detrimental to SG morphology at all
effective dilutions, so these two detergents were not tested further.
Following the removal of detergent, SGs were washed, as described
above, five times in fresh Drosophila saline.
The nonionic emulsifying polysorbates, Tween 20 and Tween
80 (Promega), were used both at 0.05%, for 30–60 s at 23 C, and
BEŇOVÁ-LISZEKOVÁ ET AL.
always diluted in Drosophila saline. After their removal, SGs were
again washed five times in fresh Drosophila saline.
To destabilize divalent, usually inorganic, cationic interactions in
which Mg2+ and/or Ca2+ ions are chiefly involved, we treated glands
with 1 mM EDTA +1 mM EGTA (Fluka and Sigma, Buchs, Switzerland,
and Steinheim, Germany, respectively) in Drosophila saline for 1 min at
23 C. After washing five times in fresh Drosophila saline, the glands
were aldehyde-fixed as described below.
All above described experiments (treatments) were at least tripli-
cated with minimum of 10 larvae (N) in each of them.
2.3 | Tissue processing for SEM
Enzymatically and chemically treated or untreated SGs were fixed in 4%
paraformaldehyde + 2% glutaraldehyde in 100 mM sodium cacodylate
buffer (pH 7.2) for 1 hr at room temperature (23 C); we usually used
100 μL of fixative per 10–20 pairs of glands. Then SGs were rinsed five
times for 5–10 min each in 100–200 μL of fresh 100 mM sodium
cacodylate (pH 7.2) at 23 C. Glands were postfixed in 0.5% osmium
tetroxide in H2O for 30 min at 23 C, and then extensively rinsed in H2O
(a minimum of six times for 10 min each). Tissues were dehydrated in an
ascending ethanol series (30, 50, 70, 96, and 100%) and then incubated
twice more in 100% ethanol, a 1:1 mixture of 100% ethanol + 100%
acetone twice and absolute acetone (thrice). During exchanges of abso-
lute ethanol or acetone, and all later time points, we took particular care
to ensure that the tissue was always present in solution and did not dry-
up. For this reason, the addition of hexamethyldisilazane (HMDS; Sigma-
Aldrich) in place of Peldri II to facilitate critical point drying (Beňo et al.,
2007) was done in two or three steps. Initially, HMDS was applied in
the presence of small remnants of acetone. After 5–10 min, the solution
was quickly removed and replaced with fresh HMDS. The samples were
left for 30 min after which HMDS was allowed to evaporate completely.
Since the evaporation of HMDS tends to generate a slight electrostatic
potential on the surface of the samples, we recommend that this last
step takes place in very clean air environment (e.g., inside a desiccator)
to minimize the attraction of dust particles.
Dried SGs were mounted on pieces of Scotch double-sided
tape on 16 or 24 mm aluminum SEM stubs while viewed under a
stereomicroscope. Samples were sputter coated for 2.5 min with
gold–palladium using the Balzers sputter coater device SCD-030 at
35–40 mA per stub and under 0.05–0.1 mbar to produce a 40–50 nm
continuous alloy layer. Samples were viewed and photographed on a
FEI Quanta FEG250 scanning electron microscope with the emission
field cathode set at 10 kV acceleration voltage. The bitmap images
obtained were processed and labeled using Adobe Photoshop or Corel
Draw software, and assembled into figures using Aldus FreeHand and
Adobe Photoshop. To keep the presentation of microscopic data uni-
form, the anterior end of SGs is always oriented to the left, and the
posterior end to the right, regardless of the magnification.
3 | RESULTS
In the course of a study where we needed to carefully document
the phenotype of Drosophila SGs during the larval-to-early pupal
3
metamorphic period, we faced the problem that an unacceptable number
of tissue samples that we processed for SEM show undesirable contami-
nation by random surface dirt (Figure 1a–d). Although the presence of
such dirt, in principle, should not interfere with the characterization of
normal and mutant developmental phenotype, the magnitude of con-
tamination rapidly increases during metamorphosis due to endogenous
histolysis of larval tissues. During that time, large amounts of sticky cel-
lular debris circulate in the hemolymph, and this debris has a strong ten-
dency to attach to the surface of many organs during their dissection. In
addition, numerous larval tissues that are preparing for programmed cell
death, even at times well before its executive phase, become attacked
by an increasing numbers of phagocytic hemocytes that interact with
their target tissue quite strongly (Figure 1e,f), so cannot be removed or
washed away as easily as randomly attached sessile hemocytes that cir-
culate prior to metamorphosis. Thus, when SEM images from these
stages are compared to the SEM images of the same tissues dissected
from an earlier developmental period an observer gains a distinct
impression, wrongly, that they are viewing either low-quality samples,
or samples that have been processed poorly. This contributes to
confusion in interpreting otherwise valid results. To avoid this type
of misapprehension and to minimize unwanted contamination,
we undertook series of simple but laborious experiments in which we
tested multiple enzymes, detergents, emulsifiers, and chelating agents
(aminopolycarboxylic acids) for their ability to remove surface dirt from
the SGs during the metamorphic period of Drosophila development.
A fat body adheres to the outside lateral (peripherolateral) side of
each SG and extends from its posterior end. We tested a panel of
enzymes that are frequently used in the preparation of primary cell
cultures from isolated tissues—trypsin, chymotrypsin, elastase, colla-
genase, pronase, proteinase K, and dispase—for their ability to digest
away fat body fragments that adhere to the SGs after their dis-
section with fine forceps. We evaluated whether a very short incuba-
tion with individual fast-acting enzymes could facilitate the removal of
fat body fragments. We optimized digestion conditions by testing
each protease over a range of active concentrations, but present here
only those we found most useful. Trypsin was used as 0.005%, chy-
motrypsin as 0.0025%, proteinase K as 5 μg/mL, elastase as 0.005%,
collagenase as 0.15 mg/mL, pronase as 25 μg/mL, and dispase as
0.01 mg/mL. When one or two enzymes were used, the digestion
time was 2 min, whereas if three or more (even all seven) enzymes
were used, the digestion time was limited 1 min to prevent overly
aggressive proteolytic action.
Dissected but untreated late larval or prepupal SGs of Drosophila
often are contaminated with adherent dirt. This can be fragments of fat
body that were unable to be removed without simultaneously damag-
ing the SGs themselves, sessile hemocytes, or a variety of unidentifiable
cellular debris that was liberated during the dissection procedure from
other tissues (Figure 1a–c). Simple, repeated rinsing of the dissected
SGs with solutions containing detergent (Figure 1d) or a combination of
detergent and emulsifiers (Figure 1e) can release some of the adhering
material, but does not remove the fat body or more strongly adhering
material. If detergents and emulsifiers are followed by few brief washes
in a mixture of EDTA and EGTA, the sessile hemocytes, but not the fat
body, are released (Figure 1f).
4 BEŇOVÁ-LISZEKOVÁ ET AL.

FIGURE 1 Salivary glands (SGs) fixed and prepared for scanning electron microscopy without any treatment. (a) A view of an entire dissected late
larval SG with a large piece of attached fat body (FB). (b) Enlarged posterior end of the SG dissected from a white prepupal stage showing the
adherence of a large piece of remaining FB and various dirt nonspecifically attached to the gland's surface (white arrows). (c) Detailed view of SG
surface contamination by various dirt deriving either from hemolymph circulation or the dissection process (white arrows). (d) Action of
detergents (Triton X-100 + sarkosyl + Na-deoxycholate) on dissected SG does not remove FB or other strongly adhering debris. (e) Although a
combination of detergents and emulsifiers (Tweens 20 and 80) can improve desired image of the SG, they are unable to remove adherent FB. (f)
The surface of the larval or prepupal SGs can be made free of sessile hemocytes when treatment with detergents and emulsifiers is followed by a
few brief washes in a mixture of EDTA + EGTA

Since we wanted to minimize the time glands were unfixed, we
sought to identify conditions that would allow for the shortest
enzyme incubation time to efficiently remove the adherent fat body
fragments. Depending on which enzyme or combination of enzymes is
used, the fat body can be removed very efficiently using a 1–2 min
digestion (Figure 2a). However, the fat body is only partially removed
when multiple enzymes are used in combination, each at the same con-
centration that was used in individual-enzyme digestions, for shorter
incubations. Figure 2b shows an example of the result obtained from a
30-s incubation with combination of trypsin, proteinase K, and collage-
nase; Figure 2c shows an example of the result of using a chymotrypsin,
elastase, and pronase. Conversely, using trypsin alone for 2 min at 25%
of what we found to be a very effective concentration (0.00125%),
resulted in quite weak removal of the fat body (Figure 2d). Similarly,
combining three diluted enzymes (0.00125% trypsin + 1.25 μg/mL
proteinase K + 0.00125% elastase) for 2 min digested more of the
adhering fat body than a diluted individual enzyme but not all
(Figure 2e). Treatment for 2 min with a solution containing dilutions of
all seven enzymes (0.00125% trypsin + 0.000625% chymotrypsin +
1.25 μg/mL proteinase K + 0.00125% elastase + 0.0375 mg/mL colla-
genase + 6.25 μg/mL pronase + 0.0025 mg/mL dispase) is only slightly
more effective (Figure 2f).
We found that digestion time relative to enzyme concentration is
critical to obtain consistent high-quality results. It is both easy and
detrimental to overdigest the SGs with enzymes, whether by increas-
ing their concentration or by extending the digestion time. Both result
in highly evident tissue damage. Figure 3a shows and example of the
result when SGs are incubated with seven proteases for 1 min at dou-
ble their optimal concentration (0.01% trypsin, 0.005% chymotrypsin,
10 μg/mL proteinase K, 0.01% elastase, 0.3 mg/mL collagenase,
50 μg/mL pronase, and 0.02 mg/mL dispase). Even a 3-min digestion
of SGs at these increased concentrations leads to the near-complete
removal of the basal lamina leaving “naked” gland cells (Figure 3b).
Very similar effects were observed when just three enzymes were
used for 4 min at an increased concentration (0.01% trypsin, 10 μg/mL
proteinase K, and 0.3 mg/mL collagenase; Figure 3c). Even a longer
digestion with a single enzyme at the optimal concentration that was
effective for a 2 min incubation can have devastating effects to the
BEŇOVÁ-LISZEKOVÁ ET AL. 5

FIGURE 2 Treatment of salivary glands (SGs) with various proteases. (a) The fat body (FB) is efficiently removed from dissected SGs using a
combination of chymotrypsin, proteinase K, and elastase for 2 min. (b) The FB is only partly removed by treating with a combination of trypsin,
proteinase K, and collagenase for 30 s. (c) Treatment with chymotrypsin, elastase, and pronase for 30 s. (d) Treatment with trypsin for 2 min at
25% of its effective concentration (0.00125%) does not remove the FB. (e) Treatment with a 0.00125% trypsin, 1.25 μg/mL proteinase K, and
0.00125% elastase for 2 min digests the FB more efficiently, albeit not completely. (f) The FB is incompletely digested following a 2-min
treatment with a combination of seven enzymes, each at a lower than standard dilution (0.00125% trypsin + 0.000625%
chymotrypsin + 1.25 μg/mL proteinase K + 0.00125% elastase + 0.0375 mg/mL collagenase + 6.25 μg/mL pronase + 0.0025 mg/mL dispase)

SG basal lamina (e.g., 5 min with proteinase K at 5 μg/mL (Figure 3d).
Conversely, even incubation with a diluted concentration of enzyme
(proteinase K, 1 μg/mL) for the same longer time results in partial or
local digestion of the gland surface (Figure 3e). We asked whether
these observations made using proteinase K were representative of all
of the enzymes. When the concentration of trypsin is from what we
found to be an optimal concentration (0.01%), digestion for 3 min will
cause large pieces of the lamina along with the plasma membrane to
peel off from the gland surface, however, it will not remove entire
basal lamina (Figure 3f). Combination of collagenase and elastase at
their individual optimal concentrations (0.15 mg/mL and 0.005%,
respectively) for an extended time of 5 min will introduce several deep
but relatively narrow local openings into basal lamina (Figure 3g). Even
extending the enzymatic incubation time from 2 to 3 min, without
changing the concentration of enzyme, produced noticeable effects.
By 3 min, chymotrypsin (Figure 3h) and pronase (Figure 3i) disrupt
cell-to-cell contacts at the level of basal lamina (Figure 3h), while colla-
genase starts to produce randomly distributed holes into the basal
lamina without any clear effect on cell morphology (Figure 3j).
Based on these findings, we evaluated the effects of the enzymes
on the removal of different types of dirt and in the context of other
treatments using the concentrations of enzymes described in Section 2,
applying them to the dissected SGs for either 1 or 1.5 min. Treatment
with protease significantly aided in the removal of the fat body, but did
not help remove or prevent other types of contamination; in contrast, it
appears to make such contamination worse. Close examination of the
surface of larval and prepupal SGs indicated that it can enhance the
adherence of random pieces of digested fat body, material of other tis-
sues that is circulating in the hemolymph, sessile hemocytes, or even
alimentary symbionts released from gut tissue ruptured during dissec-
tion. The adherence of these materials was resistant to intense and
repeated rinsing of the SGs with Ringer or phosphate buffered saline
(PBS) after enzymatic digestion (Figure 4a,b). Thus, although enzymatic
treatment was required to remove fat body fragments from the SGs, it
resulted in unwanted contamination. To remove this contamination, we
evaluated several chemical treatments, starting with three detergents:
Triton X-100, sarkosyl, and sodium deoxycholate. We evaluated a wide
range of concentrations at a set incubation time of 2 min at ambient
temperature (23 C). We concluded that optimal concentrations were
0.1% for Triton X-100, 0.05% for sarkosyl, and 0.001% for sodium
deoxycholate. While each acting separately can remove a significant
portion of the larger-sized surface dirt (Figure 4c–e), a mixture of all
6 BEŇOVÁ-LISZEKOVÁ ET AL.
three acts most efficiently (Figure 4f). Using them at higher concentra-
tions or for prolonged treatment times did not help further to clean
SGs surface from contaminants, however, and resulted in adverse
effects on the integrity of the SG tissue. Under either of these condi-
tions, their appeared to be deeper penetration and larger openings
inside of the cell membrane became apparent (Figures 4g,h). This
suggested that the removal of dirt and contamination, notably that
below 3 μm in average size, required a different type of treatment than
that of using the “big hammer” of detergents.
FIGURE 3 Legend on next page.
To this end, we used two nonionic emulsifying polysorbates or
emulsifiers, Tween 20 and Tween 80, reasoning that these would
further modify the surface tension on the SGs, thereby releasing
smaller-sized dirt. Initially, we evaluated 0.01% Tween 20 for 60 s
(Figure 5a), 0.05% Tween 20 for 30 s (Figure 5b), and 0.05% Tween
20 for 60 s (Figure 5c). There was little difference between effects
of 0.01% Tween 20 for 60 s and 0.05% Tween 20 for 30 s, but more
efficient action was seen for 0.05% Tween 20 for 60 s—this
removed all larger clumps of surface dirt. Then, we evaluated the
BEŇOVÁ-LISZEKOVÁ ET AL. 7

application of 0.01% Tween 80 for 60 s (Figure 5d), 0.05% Tween
80 for 30 s (Figure 5e), and 0.05% Tween 80 for 60 s (Figure 5f).
Both the 0.01% Tween 80 applied for 60 s and the 0.05% Tween
80 applied for 30 s had very similar effects as identically used Tween
20. However, 0.05% Tween 80 applied for 60 s significantly facili-
tated the release of clumped contaminants from the SG surface.
Finally, we evaluated the application of 0.05% Tween 20 + 0.05%
Tween 80 for 30 s (Figure 5g), or for 60 s (Figure 5h). Both facili-
tated the removal of small-sized 0.1–0.5 μm dirt which mostly aggre-
gated at randomly scattered locations into 2–5 μm clumps. Longer
treatment (60 s) was visibly more efficient (Figure 5h), and could be
considered to be sufficient for providing a surface free of
surface dirt.
When all of these treatments are applied in the sequence
described here, they can greatly help to routinely produce high-quality
SEM images of Drosophila SG tissue. However, actively adhering
hemocytes are mostly not affected by this protocol. Hemocytes are
usually sitting individually (Figure 6b) or in small groups (Figure 6a),
and randomly scattered on the surface of the SG cells. On one hand,
this may be acceptable, as it allows for functional studies of the inter-
actions between immune blood cells with SGs or other epithelial tis-
sues. On the other hand, for our purposes, we wanted to routinely
prepare hemocyte-free tissues. So, we evaluated whether reducing
divalent cationic interactions can minimize the unwanted attachment
of hemocytes. For this, we evaluated whether aminopolycarboxylate-
based chelating agents, EDTA and EGTA, release these cells from the
SG surface. We tested three different concentrations of each,
100 μM, 1 mM, and 10 mM, and established that 100 μM was not
fully efficient, whereas 1 mM was as sufficient as 10 mM to obtain a
hemocyte-free SG surface. So, in all subsequent operations, we
worked with a 1 mM concentration of both chelating agents. When
tested individually, both EDTA and EGTA showed a strong potential
to deplete sessile hemocytes if applied for 30 s (Figure 6c,d). When
applied together as an equal mixture of two 1 mM solutions either for
15 s (Figure 6e) or 30 s (Figure 6f), we saw no discernable difference.
We interpret this to indicate that mixture of two chelating agents and
their simultaneous action even for a doubled time has no adverse
effect on SG morphology.
4 | DI SCU SSION
Preparing reproducible and good-quality SEM samples from soft tis-
sues of some experimental organisms presents a constant challenge.
As we found in D. melanogaster, one of the most commonly used
genetic model organisms, the main problem for routinely obtaining
high-quality of SEM images is the irregular contamination of the tissue
surface with adherent dirt. In metamorphic stages, the majority of the
adherent material comes from the internal circulation of the organism
and that unavoidably generated by the dissecting procedure itself. To
use SEM as a routine method for the detailed morphological analysis
of developing tissues and organs, we devised a quick protocol that
dramatically increases the chance of reproducibly preparing high-
quality samples. It first removes coarse pieces of adherent fat body,
then cleans the surface of mid- and small-sized dirt including the
removal of tiny sticky contaminants, finally ending with a treatment to
detach sessile hemocytes.
Although each of the treatments has the overall effect of making
the soft SG tissue less contaminated with unwanted debris, each step is
pivotal to remove a specific category of dirt: (1) proteases, either indi-
vidually or as a mixture, digest and remove adherent fat body, (2) deter-
gents facilitate the removal of fat body remnants that are not washed
away by repeated rinses in buffer, (3) nonionic emulsifying polysorbate
emulsifiers detach numerous small and nonspecific dirt fragments asso-
ciated with the surface of the proteolytically digested and detergent-
treated SGs, and finally (4) highly efficient aminopolycarboxylate-based
chelating agents liberate sessile hemocytes from the surface of SGs.
These four key treatments must be used sequentially in the order
described here to obtain desired effects. It is not only that the applica-
tion of chelating agents or detergents can diminish or completely inhibit
the activity of some enzymes, but also that these agents must be used
after enzymatic treatment to clean all of the dirt generated by digestion
itself. If the order was to be reversed, the amount of contamination
would be drastically increased.
Our initial idea that proteolytic enzymes could be helpful to digest
the portion of the fat body that adhered to the SGs and mechanically
unremovable was correct. We considered using these enzymes based
on their wide application in tissue dissociation protocols used to

FIGURE 3 Enzymatic overdigestion of a tissue. (a) Overall view of the larval salivary gland (SG) treated with all seven proteases for 1 min at
double the standard concentration (0.01% trypsin, 0.005% chymotrypsin, 10 μg/mL proteinase K, 0.01% elastase, 0.3 mg/mL collagenase,
50 μg/mL pronase, and 0.02 mg/mL dispase). (b) a 3-min digestion of the SG at an increased concentration of all seven enzymes removes the
basal lamina almost completely, leaving SG cells uncovered. (c) Similar strong digestion effects are observed at an increased concentration if just
three enzymes are used (0.01% trypsin + 10 μg/mL proteinase K + 0.3 mg/mL collagenase) for 4 min. There is nonspecific adherence of sticky fat
globules that were released from digested fat body (FB) on the surface of the gland. (d) Treatment of the SG with single enzyme, proteinase K, at
the standard concentration (5 μg/mL) for 5 min reveals strong effects on the basal lamina and cell surface (white arrow). (e) When the same
enzyme, proteinase K, is applied for 5 min at a lower concentration (1 μg/mL), there is local digestion of the gland surface (white arrow). (f)
Incubation with twofold more than standard concentration for 3 min with trypsin (0.01%) causes large pieces of lamina along with the plasma
membrane to peel off from the gland surface, but does not entirely remove basal lamina or FB. (g) The combination of collagenase and elastase at
standard concentrations (0.15 mg/mL and 0.005%, respectively) digesting for a prolonged time (5 min) introduces several deep but relatively
narrow local openings into the basal lamina (white arrow). (h) A slightly longer than standard (3 min) overdigestion of SG with the standard
concentration of chymotrypsin (0.0025%) reveals that this enzyme acts primarily on the cell-to-cell contacts at the level of basal lamina (black
arrowheads) although it can digest also any place on the cell surface (white arrow). (i) A quite similar type of action on cell-to-cell contacts is
observed also for pronase at its standard working concentration if digestion is allowed to proceed for 3 min (white arrowheads). (j) In contrast,
digestion for 3 min with collagenase alone (0.15 mg/mL) reveals that it produces randomly distributed holes into the basal lamina without any
clear preference for cell topology
8 BEŇOVÁ-LISZEKOVÁ ET AL.

FIGURE 4 Clearing activity of detergents. (a) A larval salivary gland (SG) is cleared of fat body remnants after enzymatic digestion but has numerous
fragments of dirt (black arrows) that are resistant to enzymatic digestion and repeated rinsing with PBS buffer or saline. (b) Detailed view of another
identically treated SG showing large fragments of adhering dirt that were released during dissection and enzyme digestion (white arrows). White
arrowhead points to sessile hemocytes. (c) About 0.1% Triton X-100 specifically cleans the prepupal SG of most of the dirt (black arrows). (d) About
0.05% sarkosyl is slightly less efficient than Triton on larger pieces of dirt but more potent for releasing smaller pieces of dirt. (e) About 0.001%
sodium deoxycholate appears to be quite efficient at releasing both larger and smaller pieces of dirt. (f) When used in combination, the three
detergents together show a very high efficiency of surface cleaning, here documented on late larval SG. (g) Detergent overtreatment: using
detergents at a higher concentration or for prolonged treatment failed to clean the SG surface from contaminants, with adverse effects. (h) Detailed
view of an overtreatment showing that the deeper penetration of detergents causes larger holes (openings) inside cell membrane

obtain primary cell cultures from various animal, mostly mammalian,
tissues and organs (Edgar & Goldstein, 2012; Freshney, 2010; Gedye &
Ailles, 2013; Kruse & Patterson, 1973; Kunz-Schughart & Mueller-
Kleiser, 2000; Li, 1998; Lincoln & Gabridget, 1998; Palomares,
Estrada-Mondaca, & Ramirez, 2006; Sinha & Kumar, 2008; Vlak, de
Gooijer, Tramper, & Milzernburger, 2002; White & White, 1997; and
many useful recommendations in tissue dissociation manuals from
Worthington, Stem Cell Technologies, Thermo-Fisher Corp.). We opti-
mized digestion conditions for each protease by testing a wide range
of active concentrations. We usually started with a 10- to 500-fold
higher concentration than recommended for each enzyme's use in tis-
sue dissociation, testing decadic downward dilutions until we were
unable to observe any effect of digestion. Then, we went back and
used the lowest acceptable but still fast-acting dilution—this is the
recommended concentration in this article. However, it is important
to emphasize that successful result of enzymatic treatment that we
observe most probably reflects the fact that the contacts between
these two different organs, for example, the contacts between the SG
and a fat body, are digested more readily than the basement mem-
brane of either individual organ. In particular, it may reflect the close
BEŇOVÁ-LISZEKOVÁ ET AL. 9

FIGURE 5 Clearing activity of emulsifying polysorbates. (a) About 0.01% Tween 20 for 60 s and (b) 0.05% Tween 20 for 30 s both were unable
to remove all tiny surface contaminants (black and white arrows). (c) About 0.05% Tween 20 for 60 s was more efficient, as less and smaller dirt
was left on the salivary gland (SG) surface (black arrows). (d) About 0.01% Tween 80 for 60 s and (e) 0.05% Tween 80 for 30 s show very similar
effects, leaving fewer but slightly larger pieces of dirt on the SG surface (white and black arrows) than Tween 20. (f) About 0.05% Tween 80 for
60 s left fewer pieces of SG surface dirt (white arrows). (g) When used together, 0.05% Tween 20 and 0.05% Tween 80 for 30 s or (h) for 60 s
produced a highly cleaned SG surface with minimal or no dirt

packing of cells within epithelial tissue such as the Drosophila SGs,
which have very little intercellular material between them. The
extremely tight bond between adjacent cells, which includes tight
junctions and zonula adherens, makes their dissociation by any of the
proteases we tested more difficult.
It is also useful to consider in this context that the fat body at the
onset of metamorphosis is committed for disintegration (Aguila, Suszko,
Gibbs, & Hoshizaki, 2007; Hoshizaki, 2005; Liu et al., 2009; Nelliot,
Bond, & Hoshizaki, 2006; Postlethwait & Jones, 1978). Individual cells
of disintegrated larval fat body will survive throughout entire pupal
development. Only 1 or 2 days following the eclosion of the adult fly
will they be completely histolyzed (Richard, Arnim, & Gilbert, 1993;
Zheng, Yang, & Xi, 2016). The process by which the fat body is
remodeled and dissociated is coordinated by the same hormonal con-
trol that is associated with metamorphosis. It is mediated, among other
means, by the matrix metalloproteases Mmp1 and Mmp2 (Bond et al.,
2011; Jia et al., 2017; Jia, Liu, Liu, & Li, 2014; Liu, Jia, Tettamanti, & Li,
2013; Page-McCaw, 2008). Thus, the endogenous action of these
metalloproteases acts as supportive pretreatment that facilitates the
subsequent digestion of the fat body by exogenously supplemented
proteases. This also contributes to its easier detachment from the SG
proper. Moreover, this idea is supported by our observation that treat-
ment with either one individual protease or a group of seven proteases,
all of which attack the fat body efficiently, do not detach sessile
10 BEŇOVÁ-LISZEKOVÁ ET AL.

FIGURE 6 Effects of aminopolycarboxylate-based chelating agents on removal of sessile hemocytes. (a) A small group of hemocytes (white
arrow) on the surface of the larval salivary gland (SG; T = tracheal trunk). (b) Randomly scattered individual hemocytes (white arrows) sitting on
the surface of early prepupal SG (T = tracheal trunk). (c) Treatment with 1 mM EDTA for 30 s or (d) 1 mM EGTA for 30 s are capable of depleting
sessile hemocytes completely from the SG surface. (e) Treatment for 15 s with an equal mixture of 1 mM EDTA and 1 mM EGTA is as efficient in
detaching sessile hemocytes from the tissue as (f) equivalent treatment for 30 s

hemocytes or affect their integrity. Hemocytes are not known to have
increased Mmp1 and Mmp2 matrix metalloprotease activity during this
period (Chintapalli, Wang, & Dow, 2007; Graveley et al., 2011; Robin-
son, Herzyk, Dow, & Leader, 2013), and therefore are not as sensitive
to enzymatic treatments as fat body tissue.
We observed some minor differences in the action of individual
enzymes with respect to the efficient digestion of adhering fat body tis-
sue. These differences would likely to be more apparent if we tested
additional enzyme dilutions for longer incubation periods. However,
our goal was to devise a short protocol that can be performed quickly,
keeping the time between dissection and fixation to a minimum while
still routinely providing high-quality SEM results. The anticipated differ-
ences in the action of enzymes principally stems from two major fac-
tors: (1) the amino acid sequence recognized by the particular enzyme
(e.g., Pro-X-Gly-Pro for collagenase I, peptide bonds involving the car-
boxyl group of the basic amino acids, such as arginine and lysine for
trypsin, peptide bonds adjacent to neutral amino acids for elastase, pep-
tide bonds involving the aromatic amino acids tyrosine, phenylalanine,
and tryptophan for chymotrypsin, or N-terminal peptide bonds of non-
polar amino acids for dispase, etc.; where X is any neutral amino acid),
and (2) availability of these recognition sequences in the substrate
(largely extracellular matrix of SGs and fat body; Bergmeyer, 1983;
Copeland, 2000; Haralson & Hassell, 1995; Helgason & Miller, 2013;
Marangoni, 2002; Passonneau & Lowry, 1993; Schomberg & Salzmann,
1991). In addition, many of these proteases exhibit also some esterase
and amidase activity which potentially could have additive effects if
they act on posttranslational modifications (Burrell, 1993; Cooper,
1989; Freshney, 2010; Toone, 2006; Wold & Moldave, 1984). In spite
of variability among these factors, the differences in action of individual
enzymes for the successful removal of the fat body seem to be negligi-
ble. The pragmatically important conclusion is that any combination of
three or four enzymes or even all seven enzymes appears to be more
efficient than action of a single protease.
In the SEM images of this article, there is variation in the gross or
overall morphology of individual glands, including their surface texture
and cell shape. These differences between glands are not due to enzy-
matic or chemical treatment of the glands. The varying morphologies
arise because the SGs were dissected from different developmental
stages during late larval and early prepupal period. The differences
reflect the initiation of metamorphosis, and represent developmen-
tally linked morphological changes that will be described in a separate
paper. While this lack of uniformity might seem problematic, our
results using tissue from different developmental stages document
that the protocol described here can be used without modification
BEŇOVÁ-LISZEKOVÁ ET AL.
and independent of developmental stage. In addition, simple testing
of various incubation periods and/or varying concentration of
enzymes can easily help to modify described protocol to be applied
not only to different Drosophila tissues as brain central nerve system,
alimentary tract, imaginal discs, but also to non-drosophilid or on-
dipteran insect species or even broader representatives of arthropods.
ACKNOWLEDGMENTS
We would like to thank Bruce A. Chase, University of Nebraska, Omaha,
for critical reading and commenting on the manuscript. This work was
supported, in part, by VEGA 2/0109/13, VEGA 2/0103/17, by NATO
grants CRG-972173 and LST.CLG-977559, by MVTS-32060600/
EC-INSTRUCT-FP7-211252 grant, by COST ENBA-CA15216 grant,
EEA-Norwegian FM SK-0086 grant, and APVV-16-0219 grant to R.F.
ORCID
Robert Farkaš https://orcid.org/0000-0002-9138-7567
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SUPPOR TI NG I NFORMATION
Additional supporting information may be found online in the
Supporting Information section at the end of this article.
How to cite this article: Beňová-Liszeková D, Beňo M,
Farkaš R. A protocol for processing the delicate larval and
prepupal salivary glands of Drosophila for scanning electron
microscopy. Microsc Res Tech. 2019;1–12. https://doi.org/10.
1002/jemt.23263