BTN Protocol Guide-08

U.S. & Canada
| Antibody-Based Technologies
R&D Systems, Inc. Immunoﬂ uorescence
U.S.
Tel:
& C
(800) 343-7475
anada
R&D S
info@RnDSystems.com
ystems, Inc.
Tel: (800)343-7475
infwwwo@RnDS.rndsystems.comystems.com
E
Europe
urope
China
China
R&D Systems Europe Ltd. R&D Systems China Co., Ltd.
®
T
R&D Systems Europe Ltd.
el: +44(0)1235529449
R&D Systems China Co
Tel: (21)52380373
., Ltd.
inf
Tel:
o@RnDS
+44 (0)1235 529449U.S.
y
& C
stems
anada
.co.uk
T
inf
el:
o@RnDS
(21) 52380373
ystemsChina.com.cn
info@RnDSystems.co
R&D Systems, Inc
.uk
.
info@RnDSystemsChina.com.cn
Tel: (800)343-7475
info@RnDSystems.com
Immunofluor
Immunoﬂ
escenc
uor
e S
escence Staining of Rat Cortical Stem Cells
taining
Europe China
of Rat Cortical Stem Cells
R&D Systems Europe Ltd. R&D Systems China Co., Ltd.
A key to successful identiﬁ cation of biomolecules
®
by immuno-
Tel: +44(0)1235
>
529449
Aspirate PBS from the
Tel:
cover
(21)52380373
info@RnDSystems.co.uk info@RnDS
slips
ystemsC
and
hina.c
add distilled
om.cn
water.
A key to suc
ﬂ uorescence
cessful iden
(IF)
tifica
staining
tion of biomolec
involves
ules
careful
by immunofluo
selection
-
of speciﬁ c Carefully remove the cover slips with forceps and mount cell
rescence (
antibodies.
IF) staining

Man
invol
y
v

e
of
s c
R&D
arefu
Sy
l s
stems
election
antibodies
of specific
ha
an
ve
tib
been
odies.
extensively side down onto a drop of mounting media (R&D Sy stems,
Many of R&D
tested
Syst
for
ems
IHC/IF
antibodies
applica
ha
tions
ve been
and
e
are
xtensiv
selected
ely test
based
ed for
on a high Catalog # CTS011) on a glass slide.
IHC/IF applic
Immunofluor
signal-to-noise ratio.
ations and are
esc
The
selec
enc
follo
ted
e S
wing
based
taining
protocol is intended to be a > The slides are ready for microscopic observation.
on a high signal-to-
noise ratio
of R
starting
. The
a
f
t C
point
ollo
or
wing
tic
for
pr
al S
intracellular
otoc
t
ol
em C
IF
is intended
ells
staining of rat cortical stem cells.
For alternative protocols for cell-surface
to be a
staining
starting
and
point
IHC, please
for intracellular visit R&D Sy IF staining stems website aof rat cortict wwwal stem .RnDSystems.com/go/IHC.cells. For alternative
protocols
A
f
k
or
ey
c

ell-sur
to succ
fac
essful
e staining
identific
and
ation
IHC,
of
please
biomolec
visit
ules
R&D
b
S
y
y

st
immunofluo
ems
-
website a
re
>
t
sc
w
en
w
Ra
c
w
e (
.R
t
IF
cortical
nDS
) sta
yst
inin
ems
stem
g in
.c
vo
cells
om/go/IHC
lves c
(R&D
arefu
Sy
l se
stems
lection
Ca
of
talog
speci
#
fic
NSC001)
antibodie
were
s.
Many of
gro
R&D
wn on
Syst
sterile
ems an
cover
tibodies
slips
ha
in
ve
24-well
been e
pla
xtensiv
tes according
ely tested
to
for
the
IHC/IF
protocol
applications
outlined
and
in
ar
R&D
e selec
Sy stems
ted based
Mouse/Ra
on a
t
high
Neural
signal-t
Stem
o
Cell
> Rat cortical
-
Functional Identiﬁ
stem cells (R&D
ca
Sy
tion Kit (R&D Sy
stems Catalog #
stems Ca
NSC001)
talog # SC013).
were
grown
noise
>
on st
r
Remove media and wash cells twice with 1 mL of PBS.
a
erile
tio. The
cov
f
er
ollo
slips
wing
in
pr
24-w
otocol
ell
is
pla
int
t
ended
es accor
to
ding
be a
t
star
o the
ting point
protoc
for
> ol
in
outlined
tr
F
ac
ix
ellular
cells in with R&D
IF staining
0.5 SystmL ems
of
of
r
Mouse/R
a
4%
t cor
paraformaldehy
tical
at
st
N
em
eur
c
al
ells
St
.
em de
For
for C
alt
ell 20
erna
minutes
tive
Functional I
protocaols
den
t room temperafor
tific
cell-sur
ation Kit (R&D S
facture.e staining
ystems C
and IHC,
atalog # SC013).
please visit R&D Systems
> Remo
w
v
>
ebsit
e media and w
W
e a
ash
t w
cells
ww
ash c
.R
three
nDS
ells t
yst
times
ems
wice with 1 mL of PBS.
with
.com/go/IHC
0.5 mL of 0.1% BSA in PBS for 5
> Fix cells with
minutes each.
>
>
Rat
P
c
ermeabilize
0.5 mL of 4%
ortical stem
and
par
cells
block
aformaldeh
(R&D
cells
Systems
with
yde for
C
0.5
20
atalog
mL
minut
#
of
NSC001)
PBS
es at
containing
room temper
were
gro
10%
atur
wn on
normal
e.
sterile
donkey
cover slips
serum,
in 24-w
0.1%
ell
T
pla
riton
tes
X-100,
according
and
t
1%
o the
BSA
> Wash cells
(Blocking Buffer) for 45 minutes a
three times with 0.5 mL of 0.1%
t room tempera
BSA in PBS for
ture.
5
minutes each.
protocol outlined in R&D Systems Mouse/Rat Neural Stem Cell
Rat cortical stem cell differentiation was monitored with immunofluorescence staining. Differenti-
> Wash cells three times with 0.5 mL of 0.1% BSA in PBS for 5
ated rat neuroprogenitors were labeled with R&D Systems goat anti-rat nestin polyclonal antibody
> Permeabiliz
Func
minutes each.
e
tional I
and block
dentific
cells
a
with
tion Kit (R&D S
0.5 mL of
y
PBS
stems C
containing
atalog # SC013).
10%
(Catalog # AF2736) and stained with R&D Systems donkey anti-goat NL-493 secondary antibody
normal
>>
donk
RemoIncuba
e
v
y
e media and w
ser
te
um,
with
0.1%
300
T
ash c
rit
µl
on
of ells tprimar
X-100,
wicy e with 1 mL of PBS.
and
antibod
1% BSA
y /antibodies
(Block-
diluted in
(Catalog # NL003; green). Neurons were labeled with R&D Systems neuron-specific mouse anti--III
tubulin monoclonal antibody (TuJ1; Catalog # MAB1195) and stained with R&D Systems donkey anti-
ing B
>
uffer) f
Fix
Blocking Buffer overnight a
c
or 45 minut
ells with 0.5
es a
mL
t r
of
oom t
4% par
emper
af
t 4
ormaldeh
̊
a
C.
ture.
yde for 20 minutes at mouse NL-557 secondary antibody (Catalog # NL007; red). Nuclei were counterstained with DAPI
room t

emper
Note:
a

tur
A
e
nega
.
tive control should be performed using (blue).
> Wash cells three
> Wash cells
Blocking Buffer with no primar
times with 0.5 mL of 0.1% BSA
three times with 0.5 mL of
y antibod
in PBS
0.1% BSA
y .
for 5
minutes each.
> Wash cells three times with 0.5 mL of 0.1% BSA
in PBS
in PBS
for
for
5
5
> Incubate
minut
with minutes each.
es each.
Rat cortical stem cell differentiation was monitored with immunofluorescence staining. Differenti-
300 µl of primary antibody/antibodies diluted in
Rataed t craot rneurticaoprl sogenittem ors celwl erdie fflabeled erentiawith tion R&D waSs ystmems onigoatort eand ti-rwiatt h nestin immpolunyoclonal fluoreanstibodcencye
Block
>
ing B
>
Permeabiliz
uff
Incuba
er ov
te
ernigh
e and
with
t a
block
t 4˚ C.
300
cells
µl
with
of
0.5
a
mL
ppropria
of PBS
te
con
R&D
taining
Sy
10%
stems
> If desir
s
(C
t
atalog
ed
inin
,
g
#
c
.
oun
AF2736)
Diffe
ter-stain
re
and
ntia
stained
ted r
with
a
with
t neu
R&D
D
ro
API
p
S
r
y
o
st
g
ems
or
enit
donk
other
ors
e
we
y an
r
c
e
ti-
oun
l
goa
ab
t
e
ter-stain
NL
led
-493
wit
sec
h
ondar
R
of
&D
y
S
an
y
tibod
stem
y
s
N
normal
ote: A nega
NorthernLightsdonkey ser
TM
tive con
um,
tr

ol should be per
ﬂ uorescent 0.1% Triton secondarX-100,
formed using
and y antibod1% BSA y diluted (Block- in
g
(C
o
a
a
talog
t an
#
t
NL003;
i-rat
gr
ne
een).
stin
Neur
po
ons
ly c
w
lo
er
n
e
a
labeled
l antib
with
ody
R&D
(C
S
a
y
t
st
a
ems
log
neur
#
on-specific
AF2736
mouse
) and
an
s
ti-
ta

in
-III
ed
choic
w
tubulin
e
ith R
monoclonal
&D Sy st
an
em
tibod
s d
y
o
(T
n
uJ1;
key
C

a
a
talog
nti-
#
g
M
oa
AB1195)
t NL-4
and
93
stained
seco
with
nda
R&D
ry a
S
n
yst
ti
ems
bod
donk
y (C
ey
a
an
ta
ti-
B
ing B
lock
Blocking Buffer in the dark a
ing B
uffer) f
uff
or 45 minut
er with no primar
es at room t
y an
t room tempera
tibod
emper
y.
ature.
ture for one hour. mouse NL-557 secondary antibody (Catalog # NL007; red). Nuclei were counterstained with DAPI
log
> Wash cells three times with 0.5 mL of 0.1% BSA in PBS for 5
> Wash #
(blue).
cNells L00thr3; gee reetimes n). Neuwith rons 0.5 were mL labeof led 0.1% with RBSA &D Sin y sPBS tems fnor eu5 ron-specific
> Wash
>
cells
Wash cells three times with 0.5 mL of 0.1% BSA in PBS for 5 mouse anti-ß-III tubulin monoclonal antibody (TuJ1; Catalog # MAB1195) and
minut
minutes each.
three times with 0.5 mL of 0.1% BSA in PBS for 5 minutes each.
minutes each.
> If desired,
es each.
stained with R&D Sy stems donkey anti-mouse NL-557 secondary antibody
counter-stain with DAPI or other counter-stain of
> Wash the cells once with 0.5 mL of PBS for 5 minutes
> Incub
>
ate
Inc
wi
uba
choice.th 3
t
0
e
0
with
µl of a
300
ppro
µl
pr
of
iat
primar
e R&D S
y
y
an
ste
tibod
ms No
y/an
rthe
tibodies
rnLights
TdilutM ed in
(Catalog # NL007; red). Nuclei were counterstained with DAPI (blue).
> Aspirate PBS from the cover slips and add distilled water. Care-
fluoresc
>
en
Block
t
W
sec
ash
ing B
ondar
cells
uff
y
er o
an
three
tibod
vernigh
times
y dilut
t a
with
t 4˚ C.
ed in
0.5
Block
mL
ing
of 0.1%
Buffer
BSA
in the
in PBS for 5
fully
>
remo
If desir
ve the
ed,
co
c
v
oun
er slips
ter-stain
with
with
forceps
DAPI
and
or
moun
other
t
c
c
oun
ell side
ter-stain of
dark a

t r

oom t
minutes each.
emper
Note: A nega
ature for one hour
tive control should be per
.
formed using
down on
choic
to a
e
drop of mounting media (R&D Systems, Catalog #
> Wash
>
cells
W
thr
ash the cells once with 0.5 mL of PBS for 5 minutes.
ee
Block
times
ing B
with
uffer with no primar
0.5 mL of 0.1%
y
BSA
antibod
in PBS
y.
for 5 CTS011) on a glass slide
> Wash cells three
.
times with 0.5 mL of 0.1% BSA in PBS for 5
minut
>
es each.
Wash cells three times with 0.5 mL of 0.1% BSA in PBS for 5
> The slides ar
minut
e r
es each.
eady for microscopic observation.
minutes each. > Wash the cells once with 0.5 mL of PBS for 5 minutes
> Incubate with 300 µl of appropriate R&D Systems NorthernLights
TM
> Aspirate PBS from the cover slips and add distilled water. Care-
fluorescent secondary antibody diluted in Blocking Buffer in the fully remove the cover slips with forceps and mount cell side
dark at room temperature for one hour. down onto a drop of mounting media (R&D Systems, Catalog #
> W
Protocol
ash cells
Guide
thr
ı
ee
200
times
8
with

0.5

mL

of

0.1%

BSA

in

PBS

f

or
View
5
entire protocol
CTS011) on a glass slide
online at www.biotechniques.com/protocol
.
ı BioTechniques ı 9
minutes each. > The slides are ready for microscopic observation.
R&D Systems Protocol.indd 9 10/25/07 6:47:05 PM
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