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

D055-3
Lot 027
Page 1

MONOCLONAL ANTIBODY

Anti-M6a (Mouse) mAb
Code No.
D055-3Clone321

Subclass
Rat IgG2a κ

BACKGROUND: M6 is a membrane glycoprotein thatis abundantly expressed on central neurons in the CHS.
Two distinct murine M6 cDNA (designated M6a and M6b)whose deduced amino acid sequences are remarkably
similar to that of the major CNS myelin protein PLP/DM20
are known. PLP expression is limited to the white matter.
M6a appears in post-mitotic neurons of the brain and spinal
cord as early as embryonic day 10, and later in thehippocampus, cerebral cortex, and the granule cells of thecerebellum. In contrast, M6b is expressed at early
embryonic stage in the ventricular zone of the spinal cord,and later during development in both neurons and glia.

Quantity
100 µL

Concentration
1 mg/mL

REFERENCES:

1) Zhao, J., et al., Mol. Vis. 14, 1623-1630 (2008) [IHC]
2) Cooper, B., et al., Brain Res. 1197, 1-12 (2008)
3) Alfonso, J., et al., PNAS 102, 17196-17201 (2005)
Clone 321 is used in this reference.

SOURCE: This antibody was purified from hybridoma(clone 321) supernatant using protein G agarose. Thishybridoma was established by fusion of mouse myeloma
cell P3X with Wister rat splenocyte immunized with mouse
M6a expressing cells.

FORMULATION: 100 µg IgG in 100 µL volume of

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 mouse M6a on

Flow cytometric analysis ofMouse M6a expression in LOcells. Open histogram indicatesthe reaction of isotypic control tothe cells. Shaded histogramindicates the reaction of D055-3 tothe cells.
The descriptions of the following protocols are examples.
Each user should determine the appropriate condition.

flow cytometry.

PROTOCOLS:

APPLICATIONS:

Flow cytometric analysis for floating cells

Western blotting; Not tested
Immunoprecipitation; Not tested
Immunohistochemistry; 1-10 µg/mL
This antibody is available for the frozen section.
Immunocytochemistry ; Not tested
Flow Cytometry; 5-10 µg/mL (final concentration)

Detailed

procedure

isPROTOCOLS.

providedintheWe usually use Fisher tubes or equivalents as reaction tubes
for all step described below.

following

SPECIES CROSS REACTIVITY:
Species

HumanCells

Not tested

Reactivity on FCM

Mouse

LO, transfectant Not tested+INTENDED USE:

Rat1) 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 (5x106 cells/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 Clear Back (human Fc receptor blocking
reagent, MBL; code no. MTG-001) to the cell pellet aftertapping. Mix well and incubate for 5 minutes at room
temperature.

For research use only. Not for clinical diagnosis.

MEDICAL & BIOLOGICAL LABORATORIES CO., LTD.
URL https://ruo.mbl.co.jp e-mail support@mbl.co.jp

D055-3
Lot 027
Page 25) Add 20 µL of the primary antibody as suggested in theAPPLICATIONS diluted with the washing buffer. Mixwell and incubate for 30 minutes at room temperature.
6) Add 1 mL of the washing buffer followed bycentrifugation at 500 x g for 1 minute at room temperature.
Remove supernatant by careful aspiration.
7) Add 20 µL of 1:100 FITC conjugated anti-rat IgG (BDPharmingen: code no. 554016) diluted with the washingbuffer. Mix well and incubate for 15 minutes at room
temperature.
8) Add 1 mL of the washing buffer followed bycentrifugation at 500 x g for 1 minute at room temperature.
Remove supernatant by careful aspiration.
9) Resuspend the cells with 500 µL of the washing buffer
and analyze by a flow cytometer.
(Positive controls for Flow cytometry; LO, transfectant)Immunohistochemical staining for frozen sections

1) Animals were transcardially perfused with 4%paraformaldehyde (PFA) in 0.1 M PBS pH 7.2.
2) Brains were removed and immediately frozen in liquidnitrogen.
3) Coronal cryosections (40 µm) were washed in 0.5%
Triton X-100 in PBS and were immersed for 1 hour atroom temperature in blocking buffer (PBS containing 5%
normal rabbit serum and 0.5% Triton X-100).
4) Tip off the blocking buffer, wipe gently around eachsection and cover tissues with primary antibody diluted
with PBS as suggested in the APPLICATIONS.
5) Incubate the sections for 2 days at 4°C.
6) Wash the slides 3 times in PBS for 5 minutes each.
7) Wipe gently around each section and cover tissues with
Biotin-conjugated anti-rat Ig (DaKo JAPAN, code no.
E0464). Incubate for 4 hours at room temperature. Wash
as in step 6).
8) Wipe gently around each section and cover tissues with
Streptavidin-horseradish peroxidase. Incubate for 2 hours
at room temperature. Wash as in step 6).
9) Visualize by reacting for 10-20 minutes with substratesolution containing 7.5 mg DAB, 40 µL of 30% H2O2 in
150 mL PBS. *DAB is a suspect carcinogen and must be handledwith care. Always wear gloves.

10) Wash the slides in water for 5 minutes.
11) Counter stain in hematoxylin for 1-5 minute, wash theslides 3 times in water for 5 minutes each, and thenimmerse the slides in PBS for 5 minutes. Dehydrate byimmersing in Ethanol 3 times for 3 minutes each,followed by immersing in Xylene 3 times for 3 minuteseach.
12) Now ready for mounting.
(Positive control for Immunohistochemistry; Rat hippocampus)

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