Targeting Inhibitory Chondroitin Sulphate Proteoglycans to Promote Plasticity After Injury

Abstract

Chondroitin sulphate proteoglycans (CSPGs) are one of the major families of inhibitory extracellular matrix molecules in the central nervous system. The expression of various CSPGs is strong during early nervous system development; however, it is downregulated during maturation and up-regulated again after nervous system injury. In vivo injection of an enzyme called chondroitinase ABC, which removes the inhibitory chondroitin sulphate chains on the CSPGs, in the injured area promotes both the regeneration and plasticity of the neurons. Here, we describe the method of in vivo injection of the chondroitinase ABC into the cortex of adult rat brain and the histochemical method to assess the successfulness of the digestion.

Full text

127
Andrew J. Murray (ed.), Axon Growth and Regeneration: Methods and Protocols, Methods in Molecular Biology, vol. 1162,
DOI 10.1007/978-1-4939-0777-9_10, © Springer Science+Business Media New York 2014
Chapter 10
Targeting Inhibitory Chondroitin Sulphate Proteoglycans
to Promote Plasticity After Injury
Jessica C. F. Kwok , Janosch P. Heller , Rong-Rong Zhao ,
and James W. Fawcett
Abstract
Chondroitin sulphate proteoglycans (CSPGs) are one of the major families of inhibitory extracellular matrix
molecules in the central nervous system. The expression of various CSPGs is strong during early nervous
system development; however, it is downregulated during maturation and up-regulated again after nervous
system injury. In vivo injection of an enzyme called chondroitinase ABC, which removes the inhibitory
chondroitin sulphate chains on the CSPGs, in the injured area promotes both the regeneration and plastic-
ity of the neurons. Here, we describe the method of in vivo injection of the chondroitinase ABC into the
cortex of adult rat brain and the histochemical method to assess the successfulness of the digestion.
Key words Chondroitin sulphate proteoglycans , Perineuronal nets , Extracellular matrix ,
Chondroitinase ABC , Plasticity , Regeneration , Central nervous system , Spinal cord injury
1 Introduction
The extracellular matrix (ECM) in animals is mainly composed of
proteins (mainly fi brous proteins such as collagen, fi bronectin, and
laminin) and glycosaminoglycans (GAGs)/proteoglycans (PGs).
The general role of the ECM is to provide a scaffold for the struc-
ture of the surrounding tissue. However, the ECM also bears sig-
nalling properties and is involved in tissue development, maturation,
differentiation, cell adhesion and migration, cell survival, and tissue
homeostasis [
1 ]. In the central nervous system (CNS), ECM has
additional functions in regulating neurite extension together with
adhesion and migration of neurons. One major family of the ECM
molecules which controls the abovementioned processes in the
CNS is chondroitin sulphate proteoglycan (CSPG) [
2 , 3 ]. During
early development, CSPGs are involved in the shaping of the neu-
ronal circuitry either by acting directly as an inhibitory molecule or
through binding to different growth factors, therefore limiting
their availability [
4 ]. Apart from controlling circuitry formation

128
during development, CSPGs are also strongly up-regulated after
spinal cord injury [
5 , 6 ]. After CNS injury, adult neurons show
very limited potential to regenerate [
7 – 9 ]. This regenerative failure
is due to a loss of growth-promoting trophic factors and substrate
molecules [
10 , 11 ] and an up-regulation of growth inhibitory mol-
ecules such as CSPGs, myelin-associated molecules such as Nogo-A,
and myelin-associated glycoprotein [
5 , 8 ]. Digestion of chondroi-
tin sulphate (CS) on the CSPGs using an enzyme called chondroi-
tinase ABC (ChABC) successfully removes the CSPG inhibition
[
5 , 12 ]. Animals treated with the ChABC demonstrate signifi cant
improvement in both structural and functional recovery [
13 , 14 ].
Recently, CSPGs have also been shown to limit plasticity in the
adult nervous system [
15 – 17 ]. Apart from being found in the loose
ECM, CSPGs are also found surrounding the soma and proximal
dendrites in a structure called perineuronal net (PNN). PNN is a
layer of ECM which wraps on the neuronal surface, where synaptic
connections are made. PNNs are formed at the end of the critical
period when the neuronal system lost the majority of its plastic
ability [
17 , 18 ]. After CNS injury, a signifi cant proportion of func-
tional recovery is achieved through axonal sprouting and dendritic
remodelling [
19 – 22 ]. However, these pro-recovery events are
hampered by the up-regulated CSPGs around the injury sites and
also on the surface of the spared neurons as PNNs. The axons of
the injured neurons form dystrophic growth cones upon interac-
tion with the CSPGs [
23 ]. Once again, application of ChABC to
the relevant brain regions is able to remove this plasticity brake and
enhance recovery [
16 , 24 , 25 ]. Activation of plasticity is not just
improving recovery after CNS injury; recent studies show that
removing CSs in the PNNs may also benefi t other neurological
conditions such as dementia, memory, and learning [
26 , 27 ]. Thus,
ChABC seems to be a useful tool in enhancing regeneration and
for studying plasticity in different animal models. Here, we describe
the method of in vivo digestion of CSs in adult rat brain by ChABC
injection and the histochemical techniques used to confi rm a
successful enzymatic digestion.
2 Materials
1. Operating isofl urane (e.g., Abbott, 1–2 % in a mixture of 25 %
nitric oxide and 50 % oxygen).
2. Absolute ethanol: Dilute it with different volume of deionised
water (
D -H
2 O) for different experimental steps (such as 70 %
for disinfection).
3. Analgesics (e.g., Carprieve, Norbrook, at 5 mg/kg body
weight) and antibiotics (e.g., Terramycin, Pfi zer, 60 mg/kg).
4. Betadine.
5. Lubricant eye ointment (e.g., Allergan).
2.1 Chemicals
and Solutions
Jessica C.F. Kwok et al.

129
6. Sterile saline (e.g., Aqupharm): Warm up to 37 °C before use.
7. ChABC (protease-free form isolated from Proteus vulgaris ,
Seikagaku, Japan): Dissolve in 0.1 M sterile PBS to a fi nal con-
centration of 100 U/ml. Store the aliquots at −20 °C. On the
day of injection, thaw the aliquot on ice and keep the solution
in the icebox until injection.
8. Pentobarbitone.
1. Animals: 3-month-old adult male Sprague Dawley rats from
Charles River (UK) are used for the experiment ( see Note 1 ).
The animals are housed in groups of up to four rats under a
12-h/12-h light/dark cycle (lights on from 6 a.m. to 6 p.m.)
with room temperature (rtp) at 21 °C. Food and water are
given ad libitum. All procedures are performed in accordance
with the UK Animals (Scientifi c Procedures) Act (1986).
2. Brain atlas for stereotaxic coordinates [
28 ].
3. Stereotaxic apparatus: Stereotaxic frame for small animals
(David Kopf Instruments, UK).
4. Laboratory scale.
5. Electric hair shaver.
6. Temperature-controlled heating mat.
7. Neurosurgery materials: Sterile cotton swabs (Johnson &
Johnson, autoclaved), surgical forceps [e.g., Dumont #5 for-
ceps from Fine Science Tool (FST)], scalpel and scalpel blades,
blunt dissection scissors (FST), skin retractor (FST) or “surgi-
cal hooks” (made from clipped and bent needles), surgical drill
and small bits, Hamilton syringe (Hamilton 65459-01), verti-
cal micro-drive pump with syringe holder which can be fi xed
onto the stereotaxic frame manipulator (e.g., UMP2 pump,
World Precision Instrument), 4-0 suture needles and needle
holder (FST), 27-G needles, 1- and 2.5-mL disposable syringes
(for subcutaneous injection of the analgesic and antibiotic
solution), ligature scissors for removing sutures (FST), surgi-
cal microscope.
1. Buffered saline pre-wash (pH7.4): Dissolve 1.78 g disodium
hydrogen orthophosphate · 2H
2 O, 9 g sodium chloride in 1 l
of
D -H
2 O (pH 7.4).
2. 4 % paraformaldehyde (PFA, pH7.4): Dissolve 8 g of PFA in
200 ml of
D -H
2 O.
3. 1× phosphate-buffered saline (PBS): Dissolve 4 g of sodium
chloride, 0.1 g of potassium chloride, 0.12 g of potassium
dihydrogen phosphate, and 0.72 g of disodium hydrogen
phosphate in 500 ml of
D -H
2 O, adjust the pH to 7.4.
4. 30 % (w/v) sucrose solution: Dissolve 30 g of sucrose in
100 ml of 1× PBS.
2.2 Animals and
Stereotaxic Surgery
2.3 Perfusion
of Animals
CSPGs in Plasticity

130
1. 24-well plates.
2. Fine-hair paintbrush.
3. 1× PBS with 0.05 % sodium azide (PBS-azide): Dissolve
0.05 g of sodium azide in 100 ml of 1× PBS ( see Note 2 ).
4. TS-PBS: Prepare a 10 ml solution by mixing 0.3 ml of normal
goat serum, 0.03 ml of Triton X-100, and 9.67 ml of 1× PBS.
5. PBS-T: Prepare 100 ml of the solution by mixing 0.3 ml of
Triton X-100 with 99.7 ml of 1× PBS.
6. Biotinylated Wisteria fl oribunda agglutinin (Sigma):
Reconstitute the lectin into 1 mg/ml solution in 1× PBS.
Aliquot and store at −20 °C.
7. ABC solution: Mix one volume of solution A with one volume
of solution B in 48 volume of 1× PBS from using the reagents
in the VECTASTAIN ABC kit “Standard” (Vector Labs).
8. Tris buffered non-saline, pH 7.4 (TNS): Dissolve 6 g of
Trizma base in 1 l of
D -H
2 O. Adjust the pH to 7.4.
9. 3,3′-Diaminobenzidine (DAB) solution (Sigma): Dissolve the
tablet of DAB substrate and urea/hydrogen peroxide in 5 ml
of
D -H
2 O. Keep the solution in the dark and use within 1 h
( see Note 3 ).
10. Gelatine-coated slides: Slowly dissolve 0.5 g of gelatin in
500 ml of
D -H
2 O at 56 °C in 5–10 min for 0.1 % (w/v) gelatin
solution. Wash the glass slides in 0.1 M sodium hydroxide for
15 min, then rinse with running tap water, and drain. Put the
slides into slide holders and dip into acetone 3–4 times (per-
form this step in a fume hood). Air-dry the slides for a few
minutes in the fume hood. Then dip the slides for 3–4 times
in the 0.1 % gelatin solution. Drain the excess solution, and
leave the slides in a 37 °C oven overnight. Box up and store
the slides in a dry place.
11. Xylene.
12. DPX mounting medium.
13. Microscope.
3 Methods
1. The coordinates are given as three-dimensional ( x, y, and z )
distances (in mm) from the bregma, which is the intersection of
the coronal and sagittal sutures on the surface of the skull. The
x plane represents the medial-lateral (ML) distance, the y plane
represents the anterior–posterior (AP) distance (where a prefi x
“+” indicates the anterior end and “−“as the posterior end),
and the z plane represents the dorsal–ventral (DV) distance
(where a prefi x “+” indicates the ventral side) from the bregma.
2.4 Tissue
Sectioning and
Histochemistry
3.1 Preparation
for the Stereotaxic
Frame and Injection
Coordinates
Jessica C.F. Kwok et al.

131
2. Injection coordinates (mm) for this experiment in reference to
the bregma are:
● First injection: AP −0.7, ML −1.5, DV +1.5.
● Second injection: AP −0.7, ML −1.4, DV +1.5 ( see Notes
4 and 5 ).
1. Before the start of the surgery, ensure that all surgical tools are
clean and sterile (either by autoclaving or by immersion in
disinfectant) and are ready to use for the operation. Clean the
operating area with 70 % ethanol.
2. Ensure that the respiration and scavenging systems are work-
ing properly and the oxygen level is suffi cient to support the
entire surgery.
3. Ensure that the operating microscope and the lights are working
properly.
4. Connect the drill and the micro-drive pump to the power
supply, and check the devices to ensure proper functions. Set
up the parameters of the pump (speed: 100 nl/min, volume:
1,000 nl).
5. Fix the drill vertically onto the stereotaxic frame probe holder,
which is then secured to the x -axis of the stereotaxic frame
manipulator through the v-shape mounting clamp. Lift the
drill up to allow suffi cient space for placing the rat.
1. All surgical procedures are performed under inhalation anes-
thetic with isofl urane.
2. Inject analgesics subcutaneously 2 h before surgery ( see Note 6 ).
3. Weigh the rat before anesthesia, and check that the animal is
in a good health condition ( see Note 7 ).
4. Anesthetize the rat in an induction box, observe closely, and
wait until the rat stops moving and the breathing rate is stable
at a reduced rate.
5. Move the rat to the shaving area, and place the nose at the
opening of the isofl urane tube ( see Note 8 ).
6. Shave the skull gently, blow away the loose hairs, and clean the
skin with Betadine.
7. Move the rat to the stereotaxic surgical stage, and switch on
the isofl urane.
8. The body temperature should be maintained at 37 °C with a
heating pad during the entire surgery.
9. Apply an eye ointment including vitamin A to protect the eyes.
10. Pinch the feet with nails to make sure that the rat reaches deep
anesthesia and no refl ex is shown. Otherwise wait for longer
time until deep anesthesia is reached.
3.2 Preparation
of Surgical Tools
and Operation Area
3.3 Preparation
of Animal and
Neurosurgery
CSPGs in Plasticity

132
11. To place the rat onto the stereotaxic frame fi x one ear bar in
the apparatus, and gently position the rat’s head so that its ear
canal is securely placed onto the ear bar. Keep the head in
place, and slowly push the second ear bar to the other ear
canal. Stop immediately when feeling that a moderate pressure
is encountered. Screw tight the second ear bar to fi x the head
in position. The head should be horizontal to the ear bars and
should not move laterally.
12. Secure the rat onto an incisor (front teeth) adapter: Gently
open the lower jaw, slowly move the incisor adapter into the
mouth, and fi t the incisors at the opening of the adapter. Pull
back the adaptor slightly, and fi x it in place. Then lower the
nose clamp onto the nose only with a low pressure ( see Note 9 ).
13. Check that the tongue is in a normal position; otherwise, pull
it out gently with small blunt forceps to prevent airway block-
age ( see Note 10 ).
14. Adjust the surgical microscope, and use low magnifi cation to
obtain a clear and complete view of the top of the skull.
15. Next, make a midline skin incision with scalpel blade 10 start-
ing from caudal to the midpoint between the eyes.
16. Retract the skin sideways with surgical hooks or skin retractor
from the incision line ( see Note 11 ).
17. Measure the z coordinates of the bregma and the lambda, and
adjust the head level using the incision adaptor so that the
z coordinates are equal at the two points.
18. Measure the x and y coordinates of the bregma, calculate the
target coordinates for injection, and adjust the manipulator to
the target position accordingly.
19. Lower the drill bit until it is almost touching the skull. Increase
the microscope magnifi cation, and adjust the focus to have a
clear view of the target area.
20. Switch on the drill, and carefully drill a hole in the skull. Stop
drilling when the blood vessels on the dura become visible and
only a very thin layer of the skull is left. Remove the thin skull
layer with a pair of fi ne forceps ( see Note 12 ).
21. Repeat the above procedure for the second coordinate.
22. Prepare for the injection: Remove the probe holder together
with the drill from the manipulator and secure the micro-drive
pump onto the x -axis via the mounting clamp.
23. Load the ChABC solution into the Hamilton syringe until the
reading reaches 2 μl for each animal ( see Note 13 ).
24. Carefully fi x the syringe onto the pump, make sure that the
syringe is secured vertically, and the scale can be seen conve-
niently. Check that the pump parameters are set correctly at
100 nl/min or 6 μl/h for 10 min.
Jessica C.F. Kwok et al.

133
25. Pierce through the dura with a sharp needle tip ( see Note 14 ).
26. Bring the Hamilton needle to the correct x and y coordinates,
and lower it until it touches the dura.
27. Slowly lower the needle to the desired z coordinate, and start
the injection. Allow the pump to run for 10 min ( see Note 15 ).
28. Remove the needle slowly to avoid a backfl ow of the solution.
29. Repeat the injection procedure for the second injection.
30. Clean the opening with moist cotton swabs, and remove all
debris including loose hair or small bone fragments.
31. Suture the skin on the skull, and inject antibiotics subcutane-
ously. Inject warm saline (37 °C) subcutaneously to avoid
dehydration of the animal after surgery.
1. Keep the rat at a warm place (in a cage with blanket or in a
recovery incubator) until fully recovered. When fully recov-
ered, return the rat to a clean cage ( see Note 16 ).
2. Check the animals every day in the following week for signs of
stress including wound infl ammation, pain, wound scratching,
suture opening, and weight loss ( see Note 17 ).
3. Remove the suture 7 days after operation under brief inhala-
tion anesthesia.
1. The post-op rat is terminally anesthetized with pentobarbi-
tone, followed by perfusion with 200 ml of cold PBS pre-wash
through the heart and then 4 % PFA ( see Note 18 ).
2.
The brain is removed and post-fi xed for 24 h at 4 °C.
3. The brain is then transferred to cold 30 % sucrose for cyropro-
tection at least overnight at 4 °C before sectioning.
1. Take out the brain from the sucrose solution, and briefl y blot
it dry on a 3 mm fi lter paper.
2. Freeze down the brain on a sledge microtome platform, and
section the brain into 35 μm thick coronal or parasagittal
sections in PBS-azide ( see Note 20 ).
3. To check if the ChABC has successfully digested the CS on the
PNNs in the brain, we use a lectin called Wisteria fl oribunda
agglutinin (biotinylated, bio-WFA) which labels the CS on
the CSPGs ( see Note 21 ).
4. The brain sections are washed in 1 ml of 1× PBS for 5 min,
three times at rtp.
5. Block the sections with TS-PBS for 1 h at rtp.
6. Remove the blocking solution, add 20 μg/ml of bio-WFA
(in TS-PBS), and incubate overnight at 4 °C.
7. Remove the lectin solution, and wash the sections in PBS-T
for 15 min, three times at rtp.
3.4 Recovery from
Anesthesia
3.5 Perfusion of Rat
3.6 Tissue
Sectioning and
Histochemistry
( See Note 19 )
CSPGs in Plasticity

134
8. While washing the sections, prepare the ABC solution
(VECTASTAIN ABC kit “Standard”) by mixing 1:1:48 of
reagent A:reagent B:1× PBS, and let it stand for at least 1/2 h at
rtp for the formation of ABC complex before use ( see Note 22 ).
9. Add the premixed ABC solution to the sections, and incubate
for 1 h at rtp.
10. Rinse the sections with TNS, three times for 15 min each.
11. Prepare the DAB solution ( see Note 23 ).
12. Incubate the sections in DAB solution, and monitor the color
intensity upon the addition of DAB substrate. Stop the reac-
tion by removing the DAB solution, and wash the sections
twice with TNS for 5 min.
13. Put the sections in a big Petri dish, and mount the sections
onto gelatin-coated slides. Air-dry the sections for 1 h.
14. Dehydrate the sections through a series of ethanol solution of
increasing percentage (50, 70, 90 % and two times of 100 %
EtOH), 5 min per solution. The slides are then incubated
twice in xylene for 5 min at rtp. The slides are coverslipped
with DPX Mounting Medium ( see Note 24 ).
15. The slides are air-dried overnight at rtp in a fume hood before
documentation using a bright-fi eld microscope (Fig.
1 ).
4 Notes
1. Other rat strains and mice can also be used for the injection.
2. Sodium azide is a toxic chemical. Please follow the supplier’s
instruction and local regulation for proper handling and disposal.
3. DAB is a strong mutagen. Please wear gloves when handling
the chemical. All solutions and consumables in contact with
the DAB have to be disposed according to the supplier’s
instruction and local regulations.
4. To obtain targeting coordinates for a specifi c injection region,
subtract the atlas coordinates from the position of the animal’s
bregma in the stereotaxic apparatus. For example, if the breg-
ma’s coordinates are “AP 60, ML 15, DV 30,” then the
desired injection coordinates are “AP 60.7, ML 16.5, DV
28.5” for the fi rst injection and “AP 60.7, ML 16.4, DV 28.5”
for the second injection.
5. The coordinates used in this protocol correspond to the senso-
rimotor cortex of the hindlimb in an adult rat. We chose this
area because hindlimb functions are mostly compromised after
lesion in various spinal cord injury models. Removing inhibitory
PNNs using ChABC opens up a window of plasticity in the
selected region and may enhance the functional recovery of the
Jessica C.F. Kwok et al.

135
Fig. 1 Section collection ( a ) and staining of chondroitin sulphates with bio- WFA ( brown ) in the adult cortex 24 h
after the injection of PBS ( b , d ) or ChABC ( c , e ). ( a ) The brain sections should be collected as a six-section series
to ensure that every stack of sections covers all levels in the brain. ( b ) and ( d ) PBS injection does not remove
the WFA-positive signal in the adult cortex. Subpopulations of neurons are clearly wrapped by perineuronal nets
under high magnifi cation in ( d ). ( c ) and ( e ) 24 h after the injection of the ChABC, a clear area devoid of brown
WFA signal (both in the extracellular matrix and on the cell surface) is observed around the injected area. High-
magnifi cation image in ( e ) shows very faint remnants of perineuronal staining on neurons present in the ChABC
area, while in the area outside the digestion zone, a very clear perineuronal staining is found on the neurons.
The asterisks mark the center of the injection sites. Scale bar in ( b ) and ( c ) is 1 mm ( d ) and ( e ) is 100 μm
CSPGs in Plasticity

136
corresponding body part in response to different treatments
and rehabilitation program.
6. The analgesic is injected subcutaneously 2 h before the sur-
gery to ensure that the optimal level of analgesic effect is
reached at the point of the surgery. It can also be injected
before the start of the surgery.
7. A good health condition is important for a speedy recovery
and to avoid unnecessary complications. If an animal is ill or
under stress before the surgery, either postpone the surgery or
choose a healthier substitute for the operation.
8. It is important to make sure that the tube is fi tted properly to
the nose of the animal. Any leakage from the tube will reduce
the effi ciency of anesthesia and the leaked isofl urane will be
released to the operation area. This poses potential harm to
the researcher.
9. Proper securing of the animal to the stereotaxic frame is criti-
cal for the operation. For detailed descriptions of the proce-
dure, please refer to ref. [
29 ].
10. Closely monitor the breathing of the animal throughout the
surgery. If the animal starts to show abnormal, slow, or labored
breathing, release the animal from the stereotaxic apparatus.
Provide air without isofl urane until the breathing recovers to a
normal pattern. Check that the oxygen is at a suffi cient level.
11. In order to reveal a clear view of the skull, scientist can gently
separate the subcutaneous tissue from the skull using blunt
dissection scissors. One can further clean the connective tissue
on the bregma and lambda areas using sterile surgical blade.
To avoid the skin tissue from drying out, apply several drops
of sterile PBS to the tissue along the incision line using wet
cotton swabs.
12. To avoid drilling through the whole skull and damaging the
cortex, make sure that you only remove a small depth of the
skull each time and stop frequently to check for the depth.
13. The total injection volume is 2 μl (1 μl for each injection
site). However, when loading the solution for the injection
setup, the dead volume of the syringe should also be included
(usually ~1 μl).
14. Hold the needle at a fl at angle to avoid damaging the brain
tissue.
15. When the injection is fi nished, leave the needle in the injection
site for another 3 min before retraction. This is to ensure that the
injected solution is fully absorbed into the surrounding tissues.
16. It is helpful to place a small dish of soaked food pellets in the
cage for easy food access after surgery.
17. The image of the histology performed in this chapter is on an
adult rat brain collected at 24 h after ChABC injection.
Jessica C.F. Kwok et al.

137
Our previous study shows that a single ChABC injection in
the cortex would effectively keep the CS at a low level for at
least 10 days after injection [
30 ].
18. A good perfusion is important for minimizing background
staining from histochemistry and to maintain a nice tissue fi xa-
tion. Perfusion with PBS pre-wash is to rinse out blood cells in
the blood vessels which tend to give strong background after
staining.
19. Histology is only one of the many ways in determining the
success of ChABC digestion. One can also assay for the resid-
ual ChABC activity recovered from the injected tissues if
necessary [
31 , 32 ].
20. The sections are usually collected as six-series stacks in the fi rst
row of a 24-well plate containing PBS-azide (Fig.
1 ). This is to
ensure that each well would have sections covering the differ-
ent levels from the brain. The sections can be stored at 4 °C
for at least 3 months.
21. WFA is a lectin which demonstrates a very strong and specifi c
binding affi nity to CSs. One can also use other antibodies to
detect CS in the area, such as clone CS-56 for CSs (Sigma)
and clone 1 F6 for neurocan (DSHB).
22. If fl uorescence detection is preferred, use fl uorophore-
conjugated streptavidin instead. After incubation for 2 h at rtp,
rinse the sections in 1× PBS three times for 15 min each, before
mounting the section with anti-fading mounting medium.
23. DAB is a toxic and carcinogenic chemical. Please follow the
provider’s instruction and local laboratory practice and regula-
tion for proper handling and disposal.
24. Xylene and DPX are very volatile solution and highly fl am-
mable. Please carry out these steps in a fume hood. Xylene is a
strong organic solvent, so use anticorrosive plasticwares or
glass containers for all the procedures.
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