D371-3
Lot 004~
Page 1For Research Use Only.
Notfor use in diagnostic procedures.
Page3

Anti-P. acnes mAb (PAB antibody)
CODE No.

D371-3

CLONALITY
CLONEISOTYPE
QUANTITY

Monoclonal
TMDU2
Mouse IgM 100 L, 1 mg/mL

SOURCEIMMUNOGEN
FORMULATIONSTORAGE

Purified IgM from mouse ascites fluid
Whole bacterial lysate of P. acnes (ATCC® 11828TM)
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
1 g/mL
Immunohistochemistry 0.25 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

Human

Mouse

Rat

OtherSample

Not tested

Not tested

Not tested

P. acnes+Reactivity

Note that lipofuscin pigments cause a non-specific staining of PAB antibody.

REFERENCES

1) Isshiki T, et al. Microorganisms 9, 1668 (2021)
2) Yamaguchi T, et al. J Clin Med. 10, 983 (2021)
3) Beijer E, et al. ERJ Open Res. 7, 00486-2020. (2021)
4) Sawahata M, et al. Intern Med. 60, 777-781. (2021)
5) Okazaki F, et al. Pediatr Rheumatol Online J.19, 18 (2021)
6) Sawahata M, et al. BMC Pulm Med. 20, 288 (2020)
7) Kinoshita Y, et al. Respir Investig. 58, 421-424 (2020)
8) Takama H, et al. Acta Derm Venereol. 100, adv00182(2020)
9) Yang, G., et al., Neuropathology 38, 159-164 (2018)10) Suzuki, Y., et al. PLoS One 13, e0198518 (2018)11) Nagata, K., et al., Sci. Rep. 7, 15226 (2017)12) Goto, H., et al., Br. J. Ophthalmol. 101, 1510-1513 (2017)13) Akimoto, J., et al., J. Neurosurg. 127, 687-690 (2017)14) Asakawa, N., et al., PLoS One 7, e0179980 (2017)15) Werner, J. L., et al., Am. J. Respir. Cell Mol. Biol. 56, 121-130 (2017)16) Yamamoto, T., et al., J. Dermatol. 44, 100-101 (2017)17) Shimamura, S., et al., Mod. Rheumatol. June 20, 1-5 (2016)18) Kokuho, N., et al., Hum. Pathol. 51, 57-63 (2016)

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

e-mail support@mbl.co.jp

D371-3
Lot 004~Page 219) Omori, M., et al., J. Eur. Acad. Dermatol. Venereol. 29, 2059-2060 (2015)20) Takemori, N., et al., J. Med. Case Rep. 8, 15 (2014)21) Asahina, A., et al., J. Dermatol. 40, 501-502 (2013)22) Negi, M., et al., Mod. Pathol. 25, 1284-1297 (2012)23) Eishi, Y., Respir. Investig. 51, 56-68 (2013)24) Eishi Y., Biomed Res. Int. 2013, 935289 (2013)
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.

SDS-PAGE & Western blotting1) Suspend the sample with PBS and sonicate 3 times for 5 min.
2) Mix the sample with equal volume of Laemmli’s sample buffer.
3) Boil the sample for 3 min. and centrifuge. Load 1 L (50 g) of the sample per lane in a 1-mm-thick SDS-polyacrylamide gel(15% acrylamide) for electrophoresis.
4) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm2 for 1 hr. in a semi-dry transfer system (TransferBuffer: 25 mM Tris, 190 mM glycine, 20% Methanol). See the manufacturer's manual for precise transfer procedure.
5) To reduce nonspecific binding, soak the membrane in 10% skimmed milk (in PBS, pH 7.2) overnight at 4°C.
6) 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.)
7) Wash the membrane with PBS-T [0.05% Tween-20 in PBS] (5 min. x 3).
8) 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.
9) Wash the membrane with PBS-T (5 min. x 3).
10) 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.
11) Expose to an X-ray film in a dark room for 3 min. Develop the film as usual. The condition for exposure and development mayvary.
(Positive control for Western blotting; P. acnes)(kDa)12755037252015

Western blotting analysis of P. acnes
Lane 1: Serotype I P. acnes (ATCC® 6919TM)
Lane 2: Serotype II P. acnes (ATCC® 11828TM)
Immunoblotted with Anti-P. acnes mAb (PAB antibody) (D371-3)

D371-3
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Page 3

Immunohistochemistry for formalin fixed paraffin-embedded section
Protocol for manual method1) 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. PK7200) 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) as suggested
in the APPLICATIONS overnight at room temperature (The concentration of antibody will depend on the conditions.).
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 twice 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 PBS
for 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 controls for Immunohistochemistry; Sarcoid granuloma and hair follicle)

Sarcoid granuloma

P. acnes in hair follicle

Immunohistochemical
detection of P. acnes in humantissue
Brown: Anti-P. acnes mAb(PAB antibody) (D371-3)
Blue: Hematoxylin

Tuberculosis granuloma

Sarcoid reaction granuloma

The data were kindly provided byProf. Yoshinobu Eishi1 and Mr.
Keisuke Uchida2.
(1Department of Food and Nutrition,
Japan Women’s University, 2Divisionof Surgical Pathology, Tokyo Medical
and Dental University Hospital).

D371-3
Lot 004~
Page 4<Appendix>
The following information is the conditions when using automated slide-staining systems. This information was kindly provided
by Dr. Yoshinobu Eishi1 and Mr. Keisuke Uchida2. (1: Department of Food and Nutrition, Japan Women’s University, 2: Divisionof Surgical Pathology, Tokyo Medical and Dental University Hospital)
The following methods are introduced in the order that showed high sensitivity results in their study.
Note: Please establish appropriate condition by each user referring to this information.
Please ask each manufacturer for the details of the instruments and reagents.

A) Protocol for Leica BOND III
Working dilution; 1:500 – 1:4000
Detection system: BOND Polymer Refine Detection (Leica, code no. DS9800)
Immunostaining procedure;
Dewax
Step regent
1 Bond Dewax Solution
2 Bond Dewax Solution
3 Bond Dewax Solution
4 Alcohol
5 Alcohol
6 Alcohol7 Bond Wash Solution
8 Bond Wash Solution
9 Bond Wash Solution

Step type:
Reagent
Reagent
Reagent
Wash
Wash
Wash
Wash
Wash
Wash

Incubation time:
30sec
0sec
0sec
0sec
0sec
0sec
0sec
0sec
0sec

Temprature:
72°C
72°C
72°C
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient

Dispense type:
150μL
150μL
150μL
150μL
150μL
150μL
150μL
150μL
150μL

Pretreatment
Step regent
1 Bond ER Solution 1
2 Bond ER Solution 1
3 Bond ER Solution 1
4 Bond ER Solution 1
5 Bond Wash Solution
6 Bond Wash Solution
7 Bond Wash Solution
8 Bond Wash Solution
9 Bond Wash Solution

Step type:
Reagent
Reagent
Reagent
Reagent
Wash
Wash
Wash
Wash
Wash

Incubation time:
0sec
0sec
60min
0sec
0sec
0sec
0sec
0sec
3min

Temprature:
Ambient
Ambient
100°C
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient

Dispense type:
150μL
150μL
Intermediate
150μL
150μL
150μL
150μL
150μL
150μL

IHC staining
Step regent
1 Bond Wash Solution
2 Bond Wash Solution
3 Bond Wash Solution
4 Bond Wash Solution
5 PAB antibody
6 Bond Wash Solution
7 Bond Wash Solution
8 Bond Wash Solution
9 Bond Wash Solution
10 Bond Wash Solution
11 Post Primary
12 Bond Wash Solution13 Bond Wash Solution
14 Bond Wash Solution
15 Bond Wash Solution
16 Bond Wash Solution
17 Polymer
18 Bond Wash Solution
19 Bond Wash Solution
20 Bond Wash Solution
21 Bond Wash Solution
22 Bond Wash Solution
23 Peroxide Block
24 Bond Wash Solution
25 Bond Wash Solution

Step type:
Reagent
Wash
Wash
Wash
Reagent
Reagent
Wash
Wash
Wash
Wash
Reagent
Wash
Wash
Wash
Wash
Wash
Reagent
Wash
Wash
Wash
Wash
Wash
Reagent
Wash
Wash

Incubation time:
0sec
1min
0sec
1min
15min
0sec
1min0sec
1min0sec
8min
2min0sec
2min
2min
2min
8min
2min0sec
2min
2min
2min
5min0min1min

Temprature:
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient

Dispense type:
150μL
Open
Open
150μL
150μL
150μL
150μL
150μL
150μL
150μL
150μL
150μL
150μL
150μL
150μL
150μL
150μL
150μL
150μL
150μL
150μL
150μL
150μL
150μL
150μL

D371-3
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26
27
28
29
30
31
32
33
34
35
36
37

Bond Wash Solution
Deionized Water
Mixed DAB Refine
Mixed DAB Refine
Deionized Water
Deionized Water
Deionized WaterDeionized Water
Hematoxylin
Deionized Water
Bond Wash Solution
Deionized Water

Wash
Wash
Reagent
Reagent
Wash
Wash
Wash
Wash
Reagent
Wash
Wash
Wash

0min
1min
0min
10min
0min
1min
0min
1min
5min
0min
1min
0min

150μL
150μL
150μL
150μL
150μL
150μL
150μL
150μL
150μL
150μL
150μL
150μL

Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient
Ambient

B) Protocol for DAKO Autostainer (PT LINK and Autostainer Link 48)
Working dilution; 1:500 – 1:4000
Immunostaining procedure;
1.
Deparaffinization, dehydration and antigen retrieval treatment performed by PT LINK (DAKO) with Target Retrieval
Solution Low pH (DAKO, code no. K8005).
PAB antibody; Preheat 65°C, 95°C 20 min., cool 65°C (twice)2.
Cool down in PBS or EnVision FLEX WASH BUFFER (15 min.).
3.
Set slide glasses on the Autostainer Link 48 (DAKO).
Category

Code

Rinse
Endogenous Enzyme Block
Rinse
Primary Antibody
Rinse
Secondary Reagent
Rinse
Labelled Polymer
Rinse
Rinse
Substrate-Chromogen
RinseCounterstain
Rinse
Rinse
Rinse

SM801

SM804
SM802

SM803
SM806

NameBuffer
FLEX Peroxidase Block
Buffer
PAB antibody
BufferFLEX + Mouse (LINKER)
BufferFLEX / HRP
BufferBufferFLEX DAB+Sub-Chromo
BufferFLEX Hematoxylin
DI Water
BufferDI WaterVolume100
100100150150

Incubation
0503003002005505050(End of Protocol)

4.

Clear and mount through graded alcohol series and xylenes.

C) Protocol for Ventana BenchMark
Working dilution of PAB antibody; 1:500 – 1:4000
Detection system: OptiView
Important notes;
1.
Since mineral oil (LS) used the machine seems to inhibit PAB antibody reaction, please wash with EZ buffer and tap water
before antibody reaction.
2.
For dilution of PAB antibody (1:500 – 1:4000), please use diluent of DAKO S2022 (or PBS). The reaction of PAB antibody
is inhibited when Roche diluent or RB buffer is used.
3.
To prevent drying of the slide glasses, set the antibody reaction to room temperature.
Immunostaining procedure;
1
23

Deparaffinization [Selected]
Cell Conditioning [Selected]
CC1 [Selected]

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Page 645678910111213141516

CC1 8 Min [Selected]
CC1 16 Min [Selected]
CC1 24 Min [Selected]
CC1 32 Min [Selected]
CC1 40 Min [Selected]
CC1 48 Min [Selected]
CC1 56 Min [Selected]
CC1 64 Min [Selected]
Pre Primary Peroxidase Inhibit. [Selected]
Primary Antibody [Selected]
Primary Antibody Temperature [Selected]
Primary Antibody No Heat [Selected]
Antibody Titration [Selected]
Take the slide glasses, put into the EZ buffer.
Remove the EZ buffer after 5 min and put the EZ buffer again.
Remove the EZ buffer after 5 min and put the EZ buffer again.
Outside the machine Remove the EZ buffer after 5 min and put the EZ buffer again.
Wash with tap water for 10 min.
Dip in RB buffer.
Return the slide glasses to the machine.
Add PAB antibody (300μl or more) on the slide glasses.
17 ***** Hand Apply ( Primary Antibody ), and Incubate for [0 Hr 30 Min] *****
18 OptiView HQ Linker [Selected]
19 OptiView HQ Universal Linker [Selected]
20 Apply One Drop of OV HQ UNIV LINKR, Apply Coverslip, and Incubate for [8 Minutes]
21 OptiView HRP Multimer [Selected]
22 Counterstain [Selected]
23 Apply One Drop of [HEMATOXYLIN II] ( Counterstain ), Apply Coverslip, and Incubate for [8 Minutes]
24 Post Counterstain [Selected]
25 Apply One Drop of [BLUING REAGENT] ( Post Counterstain ), Apply Coverslip, and Incubate for [4 Minutes]* one drop is one reagent dispense
Ventana Medical Systems, Inc., 1910 Innovation Park Drive Tucson, Arizona USA
NexES v10.6220531-5