�ݺ�ߣ

Clearing
?and
?Staining
?Methods
?
By
?Jonathan
?W.
?Armbruster
?

?
Small
?specimens,
?up
?to
?about
?50cm,
?can
?have
?skeletons
?prepared
?via
?clearing
?and
?staining,
?
a
?process
?that
?renders
?the
?flesh
?invisible,
?the
?bone
?red,
?and
?the
?cartilage
?blue.
?
?The
?general
?
methods
?to
?follow
?were
?described
?by
?Potthoff
?(1984)
?and
?Taylor
?and
?van
?Dyke
?(1985).
?
?
Although
?these
?methods
?provide
?exact
?measurements
?for
?the
?various
?solutions,
?in
?practice
?
approximation
?is
?good
?enough.
?
?For
?a
?more
?detailed
?protocol
?see
?
(http://www.aquamedia.org/FileLibrary/27/Diagnostics%20-?�\%20Staining.pdf)

?
What
?you
?need
?
You
?will
?need
?certain
?chemicals
?and
?should
?make
?some
?stock
?solutions.
?
?Some
?of
?these
?can
?
be
?purchased
?cheaply,
?some
?are
?more
?expensive.
?

?
1. Hydrogen
?peroxide
?�C
?buy
?at
?a
?pharmacy,
?replace
?about
?every
?six
?months.
?
?Do
?not
?
mix
?stock
?solutions
?of
?hydrogen
?peroxide.
?
2. Potassium
?hydroxide
?�C
?mix
?a
?stock
?solution
?of
?approximately
?1%
?potassium
?
hydroxide
?by
?weight
?with
?deionized
?or
?distilled
?water.
?
?An
?easy
?approximation
?is
?to
?
put
?a
?single
?layer
?of
?potassium
?hydroxide
?chips
?in
?a
?one-?�\gallon
?jar
?lid
?and
?mix
?with
?
one
?gallon
?(4l)
?of
?water.
?
3. 95%
?ethanol.
?
4. Glacial
?acetic
?acid.
?
?
?
5. Alcian
?blue
?stain
?�C
?used
?to
?stain
?cartilage.
?
?Cheapest
?form
?available
?works
?fine
?
6. Alizarin
?red
?stain
?�C
?used
?to
?stain
?bone.
?
?A
?little
?alizarin
?goes
?a
?long
?way..
?
7. Trypsin
?�C
?a
?digestive
?enzyme.
?
?Buy
?in
?powdered
?form
?in
?the
?cheapest
?form
?available.
?
8. Glycerin
?�C
?used
?to
?house
?the
?specimens
?upon
?completion
?of
?the
?process.
?
?Again,
?use
?
the
?cheapest
?form
?available.
?
?Beauty
?supply
?stores
?sometimes
?carry
?it
?as
?it
?is
?used
?in
?
soaps.
?
?It
?is
?also
?a
?food
?additive.
?
?Glycerin
?does
?come
?in
?reagent
?grade,
?which
?is
?very
?
expensive
?and
?unnecessary.
?
9. Sodium
?borate
?�C
?used
?with
?the
?trypsin
?in
?the
?clearing
?liquid
?and
?to
?buffer
?the
?
specimens
?after
?immersion
?in
?the
?alcian
?blue
?stain.
?
?Use
?borax,
?available
?in
?the
?
laundry
?soap
?aisle
?at
?the
?grocery
?store
?�C
?much
?cheaper
?than
?reagent
?grade
?and
?just
?
as
?effective.
?
10. Thymol
?�C
?used
?to
?prevent
?fungal
?growth
?in
?specimens
?stored
?in
?glycerin.
?
?A
?little
?
goes
?a
?very
?long
?way.
?

?
Chemicals
?
?
Acetic
?Acid
? Hydrogen
?Peroxide
?
Alcian
?Blue
? Potassium
?Hydroxide
?Crystals
?
Alizarin
?Red
? Sodium
?Borate
?
Ethanol
?(95-?�\100%)
? Thymol
?Crystals
?
Glycerin
? Trypsin
?Powder
?

?
Solutions
?needed
?

1. 15%
?hydrogen
?peroxide/85%
?Potassium
?hydroxide
?�C
?mix
?as
?needed
?(I
?often
?do
?this
?
in
?the
?jar).
?
2. 30%
?acetic
?acid/70%
?ethanol/alcian
?blue
?�C
?mix
?liquids
?and
?then
?add
?enough
?alcian
?
blue
?to
?make
?a
?deep
?blue
?liquid.
?
?You
?may
?reuse
?this
?solution
?many
?times.
?
?When
?the
?
alcian
?blue
?no
?longer
?stays
?in
?solution,
?replace.
?
3. Staurated
?sodium
?borate
?�C
?add
?a
?lot
?to
?a
?jar
?of
?deionized
?water
?and
?shake
?
vigorously.
?
?Let
?sit
?for
?awhile,
?shake
?again.
?
?Make
?sure
?that
?there
?is
?still
?sodium
?
borate
?at
?the
?bottom
?of
?the
?jar
?and
?you
?have
?saturated
?sodium
?borate.
?
4. 30%
?Sodium
?Borate
?�C
?take
?your
?stock
?saturated
?sodium
?borate
?solution
?and
?mix
?it
?
with
?water
?�C
?30%
?sodium
?borate
?solution/70%
?water.
?
5. 30%
?sodium
?borate
?+
?trypsin
?�C
?mix
?when
?you
?place
?specimens
?into
?trypsin.
?
?I
?
usually
?add
?approximately
??
?teaspoon/specimen.
?
?A
?little
?less
?if
?they
?are
?small,
?a
?
little
?more
?if
?they
?are
?large.
?
6. 70%
?potassium
?hydroxide/30%
?glycerin
?�C
?mix
?by
?volume.
?
?Solution
?can
?be
?reused
?
unless
?it
?is
?too
?stained
?
7. 40%
?potassium
?hydroxide/60%
?glycerin
?�C
?as
?above.
?
8. Glycerin
?+
?thymol
?�C
?add
?a
?few
?crystals
?of
?the
?thymol
?to
?the
?glycerin
?solution.
?
?
Alternatively,
?you
?can
?dissolve
?the
?thymol
?in
?alcohol
?so
?that
?it
?is
?saturated
?and
?add
?a
?
couple
?of
?drops
?to
?the
?glycerin.
?
?You
?can
?store
?specimens
?in
?a
?glycerin/ethanol
?
solution
?(no
?greater
?than
?25%
?ethanol),
?but
?the
?specimens
?will
?not
?appear
?as
?clear.
?

?
Chemicals
?
?
10%
?Formalin
? 30%
?Sodium
?Borate
?
Ethanol
?(30%,
?70%.
?95-?�\100%)
? 30%
?Sodium
?Borate
?+
?Trypsin
?(mix
?as
?
needed)
?
15%
?Hydrogen
?Peroxide/85%
?Potassium
?
Hydroxide
?(mix
?as
?needed)
?
70%
?Potassium
?Hydroxide/30%
?Glycerin
?
30%
?Acetic
?Acid/70%
?Ethanol/Alcian
?Blue
? 40%
?Potassium
?Hydroxide/60%
?Glycerin
?
Saturated
?Sodium
?Borate
? Glycerin
?+
?Thymol
?(mix
?as
?needed)
?

?

?
Step
?1,
?Preservation:
?If
?your
?fish
?is
?not
?preserved,
?preserve
?in
?10%
?formalin
?solution
?for
?5
?
days,
?rinse
?with
?water,
?store
?in
?water
?for
?a
?couple
?of
?days,
?rinse
?again,
?and
?step
?up
?alcohol
?
(30%,
?70%)
?with
?2-?�\5
?days
?in
?each
?level
?depending
?on
?size
?(<150
?mm,
?2
?days).
?If
?specimen
?
in
?preserved
?already,
?go
?to
?step
?1a.
?
Step
?1a,
?Dissection:
?If
?the
?specimen
?is
?large,
?you
?may
?wish
?to
?filet
?one
?side
?of
?the
?
fish
?and
?remove
?the
?skin.
?
?If
?scales
?are
?large,
?you
?may
?wish
?to
?remove
?skin.
?

?
Step
?2,
?Remove
?guts:
?Remove
?the
?gastrointestinal
?tract
?and
?gonads
?of
?the
?specimen
?and
?
place
?in
?a
?vial
?that
?can
?be
?returned
?to
?the
?original
?specimen
?jar.
?

?
Step
?3,
?Dehydration:
?Place
?specimen
?in
?95%
?or
?100%
?ethanol
?(95%
?is
?much
?cheaper).
?
?
For
?small
?specimens
?(<150mm)
?leave
?in
?for
?2
?days,
?larger
?specimens
?up
?to
?a
?week.
?

?

Step
?4,
?Alcian
?Blue
?Staining
?for
?cartilage:
?Place
?specimens
?in
?30%
?acetic
?acid/70%
?
ethanol/alcian
?blue
?stain
?for
?1
?day
?(<80mm),
?1.5
?days
?(80-?�\500
?mm),
?or
?2
?days
?(>500
?mm).
?

?
Step
?5,
?Neutralization:
?Return
?alcian
?blue
?stain
?solution
?to
?stock
?bottle,
?and
?place
?
specimens
?in
?saturated
?sodium
?borate
?for
?half
?a
?day
?(<100
?mm)
?to
?2
?days
?(>100
?mm;
?you
?
may
?wish
?to
?change
?solution
?halfway
?through).
?

?
Step
?6,
?Bleaching:
?Place
?specimens
?in
?15%
?hydrogen
?peroxide/85%
?potassium
?hydroxide
?
solution.
?
?Most
?specimens
?will
?be
?depigmented
?within
?an
?hour
?(darker
?specimens
?may
?take
?
a
?little
?longer).
?
?If
?the
?specimen
?has
?little
?pigment,
?you
?may
?skip
?this
?step.
?
?DO
?NOT
?LEAVE
?
SPECIMENS
?TOO
?LONG
?IN
?THIS
?SOLUTION!
?

?
Step
?7,
?Clearing
?1:
?Place
?specimens
?in
?30%
?sodium
?borate
?solution
?and
?add
?approximately
?
?
?teaspoon
?of
?trypsin
?per
?specimen
?(if
?you
?are
?clearing
?a
?lot
?in
?a
?jar
?at
?the
?same
?time,
?you
?
can
?add
?less).
?
?You
?may
?need
?to
?change
?this
?solution
?in
?a
?couple
?of
?days
?if
?it
?turns
?blue.
?
?
Replace
?solution
?every
?7-?�\10
?days
?until
?the
?specimen
?is
?about
?60%
?clear
?(you
?can
?see
?the
?
bones
?of
?the
?vertebral
?column,
?but
?it
?isn��t
?perfectly
?clear
?yet).
?
?Keeping
?the
?specimens
?in
?
light
?may
?speed
?the
?clearing
?process.
?

?
Step
?8,
?Alizarin
?Staining
?for
?Bone:
?Add
?enough
?alizarin
?red
?dye
?to
?1%
?potassium
?
hydroxide
?solution
?to
?make
?it
?a
?deep
?reddish
?purple.
?
?This
?will
?take
?only
?a
?tiny
?amount
?of
?
alizarin
?(about
?what
?you
?can
?grab
?with
?a
?small
?forceps).
?
?Shake
?vigorously
?to
?mix.
?
?Leave
?
specimens
?in
?until
?the
?bones
?reach
?the
?level
?of
?darkness
?that
?you
?prefer
?(some
?like
?lightly
?
stained
?specimens
?and
?some
?like
?them
?almost
?purple).
?
?Usually
?1-?�\3
?days
?is
?sufficient.
?DO
?
NOT
?LEAVE
?SPECIMENS
?TOO
?LONG
?IN
?THIS
?SOLUTION!
?

?
Step
?9,
?Clearing
?2:
?Place
?specimens
?back
?into
?trypsin
?solution
?until
?suitably
?clear.
?
?What
?is
?
this?
?
?It
?is
?when
?the
?flesh
?seems
?almost
?invisible.
?
?It
?will
?not
?be
?perfectly
?clear
?because
?the
?
clarity
?in
?glycerin
?is
?part
?optical
?illusion
?(the
?refractive
?index
?of
?the
?glycerin
?and
?the
?cleared
?
flesh
?is
?about
?the
?same).
?
?Usually
?a
?week
?will
?suffice.
?

?
Step
?10,
?Stepping
?to
?Glycerin:
?This
?step
?is
?not
?necessary,
?but
?it
?helps
?to
?fix
?the
?dyes
?in
?the
?
fish
?(I
?think)
?as
?well
?as
?prepare
?the
?tissues
?for
?full
?immersion
?in
?glycerin.
?
?Place
?specimens
?
in
?stock
?70%
?potassium
?hydroxide/30%
?glycerin
?for
?2-?�\7
?days
?(depending
?on
?size)
?and
?then
?
40%
?potassium
?hydroxide/60%
?glycerin
?for
?2-?�\7
?days.
?
?Then
?place
?in
?glycerin
?with
?thymol
?
for
?final
?storage.
?

?
NOTE
?1:
?Specimens
?that
?are
?poorly
?preserved
?may
?begin
?to
?fall
?apart
?at
?any
?stage
?
during
?this
?process.
?
?If
?this
?begins
?to
?happen,
?rush
?the
?specimens
?through
?the
?
procedure
?and
?avoid
?potassium
?hydroxide!
?
?Specimens
?will
?take
?the
?dye
?in
?a
?solution
?
of
?30%
?sodium
?borate
?if
?necessary.
?

?
NOTE
?2:
?For
?extremely
?delicate
?specimens,
?you
?can
?mix
?the
?alizarin
?with
?either
?
sodium
?borate
?or
?glycerin
?for
?staining.
?
?Avoid
?potassium
?hydroxide.
?
?This
?will
?not
?
work
?as
?well,
?but
?it
?will
?work.
?

?

NOTE
?3:
?Old
?specimens
?rarely
?stain
?well
?for
?cartilage.
?
?If
?you
?wish
?to
?stain
?only
?bone,
?
skip
?steps
?3-?5.
?

?
Literature
?Cited
?

?
Potthoff,
?T.,
?1984.
?Clearing
?and
?staining
?techniques.
?In:
?Moser,
?H.G.
?(Ed.),
?Ontog
?eny
?and
?
Systematics
?of
?Fishes.
?Special
?Publication-?�\American
?Society
?of
?Ichthyolo
?gists
?and
?
Herpetologists,
?vol.
?1.
?Allen
?Press,
?Lawrence,
?KS,
?USA,
?pp.
?35�C37.
?
?

?
Taylor,
?W.R.,
?van
?Dyke,
?G.C.
?(1985)
?Revised
?procedures
?for
?staining
?and
?clearing
?small
?
fishes
?and
?other
?vertebrates
?for
?bone
?and
?cartilage
?study.
?Cybium
?9:
?107-?�\119
?

?

�ݺ�ߣ

Clearing and staining

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