For Research Use Only.
Not for use in diagnostic procedures.

D187-3
Lot 036～Page 1

MONOCLONAL ANTIBODY

Anti-Dlk (Pref-1) mAb
Code No.
D187-3

Clone
24-11

Subclass
Rat IgG1

BACKGROUND: Delta like protein (Dlk), also knownasPreadipocyte
factor-1(Pref-1)
orzonaglomerulosa-specific factor (ZOG), is an EGF-like
transmembrane protein expressed preadipocytes but not inmature adipocytes. It is highly expressed in fetal liver, theadrenal gland, and placenta, as well as someneuroendocrine tumors and small cell lung carcinomas,
where it plays a role in differentiation and proliferation.
Dlk positively and negatively regulates adipocyte
differentiation via at least four major variants (45-60 kDa)
of Dlk generated by alternatively splicing. Constitutive
expression of Dlk inhibits adipogenesis, but insulin orinsulin like growth factor-1 (IGF-1) can circumvent thisinhibition. Regulated processing of Dlk releases a 50 kDa
soluble form that was previously characterized as Fetal
Antigen-1, a protein involved in pancreatic island celldifferentiation.

SOURCE: This antibody was purified from hybridoma(clone 24-11) supernatant using protein G agarose. Thishybridoma was established by fusion of mouse myeloma
cell with rat splenocyte immunized with Dlk-Fc fusion
protein.

FORMULATION: 100 µg IgG in 100 µL volume ofQuantity
100 µL

Concentration
1 mg/mLAPPLICATIONS:

Western blotting; Not tested
Immunoprecipitation; Not tested
Immunocytochemistry; Not tested**It is reported that this antibody can be used in thisapplication in the reference number 2).
Immunohistochemistry (frozen section); 1 µg/mL
Immunohistochemistry (paraffin section); 10 µg/mL
Flow cytometry; 5-10 µg/mL
Detailed

procedure
PROTOCOLS.

isprovided

inthefollowing

REFERENCES:

1) Tan, K. S., et al., Biol. Open 4, 596-607 (2015) [IHC]
2) García-Alonso, V., et al., J. Biol. Chem. 288, 28230-28242 (2013) [IC]3) Cheung, P. F., et al., PLoS One 6, e28246 (2011) [FCM]
4) Khurana, S. and M ukhopadhyay, A., Am. J. Pathol. 173,1818-1827 (2008) [FCM]
5) Suzuki, K., et al., Gastroenterology 135, 270-281 (2008)
6) Tanimizu, N., et al., J. Cell Sci. 116, 1775-1786 (2003)
7) Kaneta, M., et al., J. Immunol. 164, 256-264 (2000)
Clone 24-11 is used in the reference number 1)-5).

PBS containing 50% glycerol, pH 7.2. No preservative iscontained.

STORAGE: This antibody solution is stable for one year
from the date of purchase when stored at -20°C.

REACTIVITY: This antibody reacts with Dlk on Flowcytometry and Immunohistochemistry.

SPECIES CROSS REACTIVITY:
Species

Human

Mouse

RatCell

Not tested

Fetal
Hepatocytes

Not tested

Reactivity on FCM

INTENDED USE:+For Research Use Only. Not for use in diagnostic procedures.

The descriptions of the following protocols are examples.
Each user should determine the appropriate condition.

MEDICAL & BIOLOGICAL LABORATORIES CO., LTD.
URL https://ruo.mbl.co.jp

e-mail support@mbl.co.jp

D187-3
Lot 036~
Page 2

PROTOCOLS:

Flow cytometric analysis for floating cells

We usually use Fisher tubes or equivalents as reaction tubes
for all steps described below.
1) Wash the cells 3 times with washing buffer [PBS
containing 2% fetal calf serum (FCS) and 0.09% NaN3].
*Azide may react with copper or lead in plumbing system toform explosive metal azides. Therefore, always flush plenty ofwater when disposing materials containing azide into drain

2) Resuspend the cells with washing buffer (5x106cells/mL).
3) Add 50 µL of the cell suspension into each tube, andcentrifuge at 500 x g for 1 minute at room temperature(20~25°C). Remove supernatant by careful aspiration.
4) Add 10 µL of normal goat serum containing 1 mg/mL
normal human IgG and 0.09% NaN3 to the cell pellet
after tapping. Mix well and incubate for 5 minutes atroom temperature.
5) Add 40 µL of the primary antibody at the concentrationof as suggested in the APPLICATIONS diluted in thewashing buffer. Mix well and incubate for 30 minutes atroom temperature.
6) Add 1 mL of the washing buffer followed bycentrifugation at 500 x g for 1 minute at roomtemperature. Remove supernatant by careful aspiration.
7) Add PE conjugated anti-rat IgG antibody diluted with thewashing buffer. Mix well and incubate for 15 minutes atroom temperature.
8) Add 1 mL of the washing buffer followed bycentrifugation at 500 x g for 1 minute at roomtemperature. Remove supernatant by careful aspiration.
9) Resuspend the cells with 500 µL of the washing buffer
and analyze by a flow cytometer.
(Positive control for Flow cytometry; Mouse fetal
hepatocytes, E14.5)

non-specific staining. Do not wash.
5) Tip off the blocking buffer, wipe gently around eachsection and cover tissues with primary antibody diluted
with PBS containing 3% normal goat serum as suggested
in the APPLICATIONS.
6) Incubate the sections for 1 hour at room temperature.
7) Wash the slides 3 times in PBS for 5 minutes each.
8) Wipe gently around each section and cover tissues with
Biotinylated secondary antibody (Vector). Incubate for
30 minutes at room temperature. Wash as in step 7).
9) Wipe gently around each section and cover tissues withVectastain® ABC Kit (Vector). Incubate for 30 minutes atroom temperature. Wash as in step 7).
10) Visualize by reacting for 10-20 minutes with substratesolution containing 7.5 mg DAB, 40 µL of 30% H2O2 in150 mL PBS. *DAB is a suspect carcinogen and must be
handled with care. Always wear gloves.
11) Wash the slides in water for 5 minutes.
12) Now ready for mounting.
(Positive control for Immunohistochemistry; Mouse fetalliver, E14)

Immunohistochemical detection of
Dlk on paraffin embedded section ofmouse fetal liver (E14.5) with D187-3.
Immunohistochemical staining for paraffin-embedded
sections

Immunohistochemical detection of
Dlk on frozen section of mouse fetalliver (E14) with D187-3.

This data was provided from Laboratory of Cell
Growth and Differentiation, IMCB, The
University of Tokyo.

Immunohistochemical staining for frozen sections1) Mouse fetal liver was embedded in OCT compound.
2) Make the frozen sections using Microtome.
3) Fix the sections with 4% paraformaldehyde in PBS.
4) Cover tissues with normal goat serum to block1) Deparaffinize the sections with Xylene 3 times for 3-5minutes each.
2) Wash the slides with Ethanol 3 times for 3-5 minuteseach.
3) Wash the slides with PBS 3 times for 3-5 minutes each.
4) Remove the slides from PBS, wipe gently around eachsection and cover tissues with blocking buffer (20 mM
HEPES, 1% BSA, 135 mM NaCl) for 5 minutes at roomtemperature (20~25°C) to block non-specific staining.
Do not wash.
5) Tip off the blocking buffer, wipe gently around eachsection and cover tissues with primary antibody diluted
with PBS containing 1% BSA as suggested in theAPPLICATIONS.
6) Incubate the sections for 1 hour at room temperature.
7) Wash the slides 3 times in PBS for 5 minutes each.
8) Wipe gently around each section and cover tissues with
Histostar (Ms + Rb) (MBL; code no. 8460). Incubate for
1 hour at room temperature.

D187-3Lot 036~
Page 39) Wash the slides 3 times in PBS for 5 minutes each.
10) Visualize by reacting for 5 minutes with Histostar DAB(MBL; code no. 8469) at room temperature. *DAB is asuspect carcinogen and must be handled with care.
Always wear gloves.
11) Wash the slides in water for 5 minutes.
12) Counter stain in hematoxylin for 1 min., wash the slides
3 times in water for 5 minutes each, and then immerse
the slides in PBS for 5 minutes.
13) Dehydrate by immersing in Ethanol 3 times for 3 minutes
each, followed by immersing in Xylene 3 times for 3
minutes each. Now ready for mounting.
(Positive control for Immunohistochemistry; Mouse fetalliver, E14.5)
For more information, please visit our web site.
https://ruo.mbl.co.jp/.

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