Anti P2RY12, Guinea Pig

P2RY12 (also known as P2Y12) is an adenosine 5‘-diphosphate (ADP) receptor and a member of the P2Y family of purinergic G protein-coupled receptors. In the central nervous system, it is specifically expressed in microglia¹⁾ but not macrophage, and is used as a homeostatic microglial marker.
The “Anti P2RY12, Guinea Pig” is a guinea pig polyclonal antibody, raised against P2RY12. It can be used to perform multiplex immunohistochemistry.

Antibody Information

* Experience with rats has been reported. For details, please see “Data”.

Protocol (Example)

1.Perfusion fixation

Perfuse with sodium citrate/PBS to remove blood, followed by perfusion with 4% PFA.

2.Post-fixation

4% PFA (-24 hours)

3.Immersion in sucrose

30% Sucrose/PBS (4℃, overnight - approximately 2 days)

4.Preparation of frozen sections

After removing sucrose, prepare frozen blocks and cut 30-μm thick sections using a cryostat.

5.Blocking

5% normal goat serum in 0.3% Triton X-100/PBS (4℃, overnight)

6.Primary antibody reaction

Anti P2RY12, Guinea pig (1:1,000), 1% normal goat serum in 0.3% Triton X-100/PBS (4℃, overnight - 2 days).

7.Washing

0.3% Triton X-100/PBS (5 minutes x 3)

8.Secondary antibody reaction

Alexa Fluor^® 488 AffiniPure Goat Anti-Guinea Pig IgG (H+L) (1:500) (Jackson ImmunoResearch, Product Number: 106-545-003), 1% normal goat serum in 0.3% Triton X-100/PBS (room temperature, 2 hours)

9.Washing

0.3% Triton X-100/PBS (5 minutes x 3)

10.Mounting

Store at 4℃ in a dark place

Performance Data

Comparison with anti-Iba1 antibody

Immunohistochemistry was performed on mouse brain (frozen sections) using anti-P2RY12 and anti-Iba1 antibodies.

Data by courtesy of
Dr. Miyata, Department of Applied Biology, Kyoto Institute of Technology

Antibodies

P2RY12
Primary Antibody: Anti P2RY12, Guinea Pig (Product Number: 011-28873, This product) 1:1,000
Secondary Antibody: Alexa Fluor^® 488 AffiniPure Goat Anti-Guinea Pig IgG (H+L) (Jackson ImmunoResearch, Product Number: 106-545-003)

Iba1
Primary Antibody: Anti Iba1, Rabbit (for Immunocytochemistry) (Product Number: 019-19741) 1:1,000
Secondary Antibody: Alexa Fluor^® 594-AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch, Product Number: 111-585-003)

[Result]
The co-localization of P2RY12 and Iba1 signals was confirmed.

Differentiating between microglia and macrophages

In the cerebral cortex and the medulla oblongata area postrema, triple staining was performed using anti-P2RY12 antibody (microglia marker), anti-Iba1 antibody (microglia/macrophage marker), and anti-laminin antibody (basement membrane marker of cerebral blood vessels), and the localization of cells was confirmed.

Data by courtesy of
Dr. Miyata, Department of Applied Biology, Kyoto Institute of Technology

Antibodies

P2RY12
Primary Antibody: Anti P2RY12, Guinea Pig (Product Number: 011-28873, This product) 1:900
Secodary Antibody: Alexa Fluor^® 488 AffiniPure Goat Anti-Guinea Pig IgG (H+L) (Jackson ImmunoResearch, Product Number: 106-545-003)

Iba1
Primary Antibody: Anti Iba1, Rabbit (for Immunocytochemistry) (Product Number: 019-19741) 1:500
Secondary Antibody: Alexa Fluor^® 594-AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch, Product Number: 111-585-003)

Laminin
Primary Antibody: Anti Laminin Antibody (Rat, Made in Dr. Miyata’s Lab) 1:200
Secondary Antibody: DyLight™ 405-conjugated AffiniPure™ Goat Anti-Rat IgG (H+L) (Jackson ImmunoResearch, Product Number: 112-475-003)

[Result]
Among the Iba1-positive cells, those that are present in the brain parenchyma are P2RY12-positive and are thought to be microglia (arrowheads). On the other hand, Iba1-positive/P2RY12-negative cells are present around the blood vessel basement membrane, and it is thought that these cells are macrophages (arrows).

Comparison with anti-Iba1 antibody and anti-TMEM119 antibody

Immunohistochemistry was performed on mouse brain and spinal cord (frozen sections) using anti-P2RY12, anti-Iba1 and anti-TMEM119 antibodies

Antibodies

P2RY12
Primary Antibody: Anti P2RY12, Guinea Pig (Product Number: 011-28873, This product) 1:1,000
Secondary Antibody: Alexa Fluor^® 647 AffiniPure Donkey Anti-Guinea Pig IgG (H+L) (Jackson ImmunoResearch, Product Number: 706-605-148)

Iba1
Primary Antibody: Anti Iba1, Goat (Product Number: 011-27991) 1:1,000
Secondary Antibody: Alexa Fluor^® 488 AffiniPure Donkey Anti-Goat IgG (H+L) (Jackson ImmunoResearch, Product Number: 705-545-147)

TMEM119
Primary Antibody: Anti-TMEM119 Antibody (Rabbit, Competitor’s Product) 1:100
Secondary Antibody: Alexa Fluor^® 594-AffiniPure Donkey Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch, Product Number: 711-585-152)

[Result]
Compared to anti-TMEM119 antibody, our anti-P2RY12 antibody staining was able to detect fine protrusion structures more clearly.

Comparison with conventional product

Immunohistochemical staining was performed on mouse brain and spinal cord (frozen sections) using our company's and competitor’s anti-P2RY12 antibodies.

Antibodies

Fujifilm Wako
Primary Antibody: Anti P2RY12, Guinea Pig (Product Number: 011-28873, This product) 1:1,000 (Antibody concentration 0.5 μg/mL)
Secondary Antibody: Alexa Fluor^® 488 AffiniPure Donkey Anti-Guinea Pig IgG (H+L) (Jackson ImmunoResearch, Product Number: 706-545-148)

Conventional product
Primary Antibody: Anti-P2RY12 Antibod (Rabbit, Competitor’s Product) 1:200 (Antibody concentration 0.5 μg/mL)
Secondary Antibody: Alexa Fluor^® 488 AffiniPure Donkey Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch, Product Number: 711-545-152)

[Result]
With the conventional product, nonspecific signals thought to be from neurons were observed (arrowheads). On the other hand, with this product, almost no nonspecific signals were observed.

Application Data

Immunohistochemistry

Mouse

Species: Mouse (C57BL/6J)
Site: Hippocampus (CA2)
Sample: Frozen section
Antibody concentration: 1:200
Notes: Blue indicates DAPI.

Data by courtesy of
Dr. I, F University

Species: Mouse
Site: Prefrontal cortex
Sample: Frozen section
Antibody concentration: 1:900
Notes: Blue indicates DAPI.

Data by courtesy of
Dr. Yu, Graduate School of Medicine, Tohoku University

Species: Mouse
Site: Cerebral cortex
Sample: Frozen section
Antibody concentration: 1:10,000
Notes: Blue indicates DAPI.

Data by courtesy of
Dr. Yamada, Faculty of Medical Sciences,
Kyushu University

Species: Mouse
Site: Hippocampus
Sample: Frozen section
Antibody concentration: 1:500

Data by courtesy of
Dr. Hashimoto,
Institute for Human Life Science,
Ochanomizu University

Species: Mouse
Site: Hippocampus
Sample: Frozen section
Antibody concentration: 1:1,000
Notes: Activated treatment (sodium citrate buffer)

Data by courtesy of
Dr. Hashimoto,
Institute for Human Life Science,
Ochanomizu University

Species: Mouse
Site: Median eminence
Sample: Frozen section
Antibody concentration: 1:1,000

Data by courtesy of
Dr. Furube,
Department of Functional Anatomy and Neuroscience,
Asahikawa Medical University

Species: Mouse
Site: Spinal cord
Sample: Frozen section
Antibody concentration: 1:2,000

Data by courtesy of
Dr. Tsuda and Dr. Kohno,
Department of Molecular and System Pharmacology,
Graduate School of Pharmaceutical Sciences,
Kyushu University

Species: Mouse
Site: Retina
Sample: Paraffin section
Antibody concentration: 1:400
Notes: Activated treatment (TE buffer, pH9.0, 3 minutes of pressure)

Data by courtesy of
Dr. Ishizuka,
Department of Pathophysiology and Metabolism,
Kawasaki Medical School

Rat

The arrowhead is thought to be a perivascular macrophage.

Species: Rat
Site: Cerebral cortex
Sample: Frozen section
Antibody concentration: 1:500
Notes: Activated treatment (pH6.0 citrate buffer, 75℃, 20 minutes)
　　　Blue indicates DAPI.

Data by courtesy of
Dr. Koizumi, Anatomy and Neurobiology,
Kyoto Prefectural University of Medicine

1. Butovsky, O. et al.: Neurosci., 17(1), 131(2014).
   Identification of a unique TGF-β–dependent molecular and functional signature in microglia
2. Keren-Shaul, H. et al.: Cell, 169(7), 1276(2017).
   A unique microglia type associated with restricting development of Alzheimer’s disease
3. Maeda, J. et al.: Brain Commun., 3(1), fcab011(2021).
   Distinct microglial response against Alzheimer's amyloid and tau pathologies characterized by P2Y12 receptor