D372-3
Lot 002~
Page 1For Research Use Only.
Not for use in diagnostic procedures.

Anti-Mycobacteria mAb (LAM antibody)
CODE No.

D372-3

CLONALITY
CLONEISOTYPE
QUANTITY

Monoclonal
TMDU3
Mouse IgM 100 L, 1 mg/mL

SOURCEIMMUNOGEN
FORMULATIONSTORAGE

Purified IgM from mouse ascites fluid
Fractionated mycobacteria by anion-exchange chromatography
PBS containing 50% Glycerol (pH 7.2). No preservative is contained.
This antibody solution is stable for one year from the date of purchase when stored at -20°C.

APPLICATIONS-CONFIRMED

Western blotting
5 g/mL
Immunohistochemistry 1 g/mL (paraffin section)
Heat treatment for paraffin embedded section: microwave oven, for 40 min. at 97°C in 10 mM citrate buffer (pH 6.2)

SPECIES CROSS REACTIVITY on WB
Species

HumanMouse

Rat

OtherSample

Not tested

Not tested

Not tested

Lipoarabinomannan (LAM)from Mycobacterium
tuberculosis Aoyama-B

Reactivity









REFERENCES

1) Yang, G., et al., Neuropathology 38, 159-164 (2018)2) Sakakibara, Y., et al., Respiration 93, 264-270 (2017)3) Iida, T., et al., J. Mycobac. Dis. 4, 142 (2014)

For more information, please visit our web site https://ruo.mbl.co.jp/.

RELATED PRODUCTS
For more information, please visit our web site https://ruo.mbl.co.jp/.
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

D372-3
Lot 002~
Page 2PaSDS-PAGE & Western blotting
ge of Laemmli’s sample buffer, then sonicate briefly (up to 10 sec.).
1) Mix the sample with equal volume2) Boil the sample for 3 min. and centrifuge. Load 20 L (10 g) of the sample per lane in a 1-mm-thick SDS-polyacrylamide
3gel (12.5% acrylamide) for electrophoresis.
3) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm 2 for 1 hr. in a semi-dry transfer system(Transfer Buffer: 25 mM Tris, 190 mM glycine, 20% MeOH). See the manufacturer's manual for precise transfer procedure.
4) To reduce nonspecific binding, soak the membrane in 10% skimmed milk (in PBS, pH 7.2) overnight at 4°C.
5) Incubate the membrane with primary antibody diluted with 1% skimmed milk (in PBS, pH 7.2) as suggested in theAPPLICATIONS for 1 hr. at room temperature. (The concentration of antibody will depend on the conditions.)
6) Wash the membrane with PBS-T [0.05% Tween-20 in PBS] (5 min. x 3 times).
7) Incubate the membrane with 1:5,000 of Anti-IgG (Mouse) pAb-HRP (MBL; code no. 330) diluted with 1% skimmed milk (inPBS, pH 7.2) for 1 hr. at room temperature.
8) Wash the membrane with PBS-T (5 min. x 3 times).
9) Wipe excess buffer on the membrane, and then incubate it with appropriate chemiluminescence reagent for 1 min. Remove
extra reagent from the membrane by dabbing with paper towel, and seal it in plastic wrap.
10) Expose to an X-ray film in a dark room for 5 min. Develop the film as usual. The condition for exposure and development
may vary.
(Positive control for Western blotting; Lipoarabinomannan from Mycobacterium tuberculosis Aoyama-B)

(kDa)
250
1501007550

Lipoarabinomannan (LAM)

372520Western blot analysis of Lipoarabinomannan from Mycobacterium tuberculosis
Aoyama-B
Immunoblotted with Anti-Mycobacteria mAb (LAM antibody) (D372-3)

D372-3
Lot 002~Page 3

Immunohistochemistry for formalin fixed paraffin-embedded section1) Deparaffinize tissue sections in Xylene 3 times for 3 min. each.
2) Immerse the slides with Ethanol 3 times for 3 min. each, then wash the slides in PBS 3 times for 3 min. each.
3) Remove the slides from PBS and heat-treat with 10 mM Citrate buffer (pH 6.2) for 40 min. at 97°C using microwave oven.
4) Let the slide cool down until at room temperature in the Citrate buffer.
5) Remove the slides from the Citrate buffer and inactivate endogenous peroxidase with 3% H2O2 in Methanol for 10 min.
6) Wash the slides with PBST [0.25% Tween-20 in PBS] 3 times for 5 min. each.
7) Incubate the sections with 2.5% normal horse serum (Vectastain Universal Elite ABC Kit, Vector Laboratories; code no.
PK-7200) for 30 min. at room temperature to block non-specific staining. Do not wash.
8) Incubate the sections with primary antibody diluted with DAKO REAL Antibody diluent (Dako; code no. S2022) assuggested in the APPLICATIONS overnight at room temperature. (The concentration of antibody will depend on theconditions.)
9) Wash the slides 3 times in PBST for 5 min. each.
10) Incubate the sections with Biotinylated anti-mouse/rabbit IgG (Vectastain Universal Elite ABC Kit) for 30 min. at roomtemperature.
11) Wash the slides 3 times in PBST for 5 min. each.
12) Incubate the sections with ABC reagent (Vectastain Universal Elite ABC Kit). Incubate for 30 min. at room temperature.
13) Wash the slides 3 times in PBST for 5 min. each.
14) Visualize by reacting for 8 min. with Histofine Simplestain DAB Solution (Nichirei Biosciences; code no. 415171). *DAB isa suspect carcinogen and must be handled with care. Always wear gloves.
15) Wash the slides 2 times in PBS for 5 min. each.
16) Counterstain in hematoxylin for 5 min., wash the slides 3 times in water for 5 min. each, and then immerse the slides in PBSfor 5 min.
17) Dehydrate by immersing in Ethanol 3 times for 5 min. each, followed by immersing in Xylene 3 times for 3 min. each. Now
ready for mounting.
(Positive control for Immunohistochemistry; Pulmonary tuberculosis lung tissue)

Pulmonary Tuberculosis

Pulmonary Tuberculosis(caseous necrosis)

Immunohistochemical detectionof Lipoarabinomannan (LAM) in
pulmonary tuberculosis lungtissue.
Sarcoid granuloma

Sarcoid reaction granuloma

Brown: Anti-Mycobacteria mAb(LAM antibody) (D372-3)
Blue: Hematoxylin
The data were kindly provided by Prof.
Yoshinobu Eishi1 and Mr. Keisuke
Uchida2.
(1Department of Human Pathology,
Tokyo Medical and Dental University
Graduate School, 2Division of Surgical
Pathology, Tokyo Medical and Dental
University Hospital).
201215-2