Serotonin-related Antibodies

5-HT_1Areceptor and 5-HT_2C receptor are G protein-coupled receptors activated by serotonin (5-HT). Both are localized mainly in the central nervous system, and their functions controlling memory, food intake, sleep, pleasure, and anxiety, etc. Anxiolytics and antipsychotics acting on these receptors have been developed, and they are attracting attention of researchers as novel targets for drug discovery.

This product is a rat monoclonal antibody established by DNA immunization and specifically recognizes native forms of 5-HT_1A receptor and 5-HT_2C receptor.

Features

• Compatible with immunohistochemical staining
• Specifically recognizes native forms of 5-HT_1Areceptor or 5-HT_2C receptor
• Rat monoclonal antibodies established by DNA immunization

Description

	Anti 5-HT1A receptor antibody	Anti 5-HT2C receptor antibody
Clone No.	4A6	6D2
Applications	Immunohistochemistry (1:100-2,000) Flow cytometry(1:100-1,000)	Immunohistochemistry (1:200-10,000) Flow cytometry(1:100-1,000)
Subclass	Rat IgG2b・κ	Rat IgG2a・κ
Cross-reactivity	Mouse ※not tested for other animal species
Antigen	Vector expressing mouse 5-HT1A receptor	Vector expressing mouse 5-HT2C receptor

DNA immunization

What is DNA immunization?

DNA immunization is a technique for generating antibodies against target proteins by using antigen proteins expressed in the animal body with expression vectors containing the target genes.

Features of DNA immunization

• Yields Antibodies specifically recognizing native forms of proteins
• Suitable for preparation of antibodies recognizing membrane proteins that were difficult to prepare by conventional methods

Principles of DNA immunization

Usage example - Immunohistochemical staining

5-HT_1Areceptor antibody

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• Localization in the cell body of prefrontal neurons was confirmed.
  This localization is consistent with the site highly expressing mRNA for 5-HT_1A.

5-HT_2C receptor

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• Localization in the cell body of prefrontal neurons was confirmed.
  This localization is consistent with the site highly expressing mRNA for 5-HT_2C.

Experimental conditions

Specimen: Prefrontal area of the brain from 10-week-old wild-type mice
Sections: 12 µm-thick frozen sections
Activation condition: Microwave treatment in a citrate buffer (pH 7.0) for 10 minutes
Antibody concentration: 1 µg/mL

Usage example - Flow cytometry

5-HT_1A receptor antibody

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• An obvious shift was observed only for cells expressing 5-HT_1A.

Anti 5-HT_2C receptor antibody

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• A clearer shift was observed for cells expressing 5-HT_2C, compared with the competitor.

Usage example - Immunohistochemical staining of the site expressing mRNA for 5-HT_1A

Experimental conditions

Specimens: Various areas of the brain from 10-week-old wild-type mice
Sections: 12 µm-thick frozen sections
Activation condition: Microwave treatment in a citrate buffer (pH 7.0) for 10 minutes
Antibody concentration: 1 µg/mL

Usage example - Immunohistochemical staining of the site expressing mRNA for 5-HT_2C receptor

Experimental conditions

Specimens: Various areas of the brain from 10-week-old wild-type mice
Sections: 12 µm-thick frozen sections
Activation condition: Microwave treatment in a citrate buffer (pH 7.0) for 10 minutes
Antibody concentration: 1 µg/mL

All immunohistochemical staining image data presented above are courtesy of Dr. Matsuda, Graduate School of Pharmaceutical Sciences, Osaka University in Japan and Dr. Takuma and Hasebe of Graduate School of Dentistry, Osaka University in Japan.

Serotonin transporter is a 12-transmembrane protein. It incorporates extracellular serotonin in the brain into the presynapse to modulate serotonin levels. With its reported involvement in sleep, fear, and anxiety, serotonin transporter has been investigated as a target of antidepressants.

Features

• Compatible with immunohistochemical staining
• Established by DNA immunization

Description

	Anti-mouse serotonin transporter, rat monoclonal antibody (R5-3-2)
Clone No.	R5-3-2
Applications	Immunohistochemistry(1：100) Flow cytometry(1：100～10000)
Subclass	Rat IgG2a・κ
Cross-reactivity	Mouse ※not tested for other animal species
Antigen	Vector expressing mouse serotonin transporter

Usage example - Immunohistochemical staining (fluorescence staining)

Immunohistochemical staining was performed for specimens from dorsal raphe nuclei (DRN) rich in serotonin neurons where expression of serotonin transporter has been reported. As a result, fluorescence signals were noted in the neuronal cell body.

Image data are courtesy of Dr. Takuma and Hasebe of Graduate School of Dentistry, Osaka University.

Protocol example

1. Wash the tissue section on the slide glass with phosphate-buffered saline containing 0.3 % Triton X-100^® (PBST).
2. Perform activation treatment in a citrate buffer (pH 6.0) with a microwave oven (10 minutes).
3. Bring the tissue section back to ambient temperature.
4. Wash the tissue section with 0.3 % PBST.
5. Block the tissue section with 5% goat serum-PBS (1 hour).
6. Incubate the tissue section with the primary antibody (this product) diluted 100-fold with 0.3 %PBST (4°C, overnight).
7. Wash the tissue section with 0.3 % PBST.
8. Incubate the tissue section with the secondary antibody (room temperature, 2 hours).
9. After washing with 0.3% PBST, mount the tissue section.

Triton X-100 is a registered trademark of Dow Chemical Company.

Usage example - Immunohistochemical staining (DAB staining)

Fluorescence signals were noted in the neuronal cell body.

Experimental conditions Samples: Frozen sections of the dorsal raphe nuclei from the brain of 10-week-old ICR mice Staining method: Enzyme-labeled antibody method (DAB staining) Activation condition: Microwave treatment in a citrate buffer (pH 6.0) for 10 minutes Antibody concentration: 1:100

Image data are courtesy of Dr. Takuma and Hasebe, Graduate School of Dentistry, Osaka University in Japan.

Protocol example

1. Wash the tissue section on the slide glass with phosphate-buffered saline containing 0.3 % Triton X-100^® (PBST).
2. Perform activation treatment in a citrate buffer (pH 6.0) with a microwave oven (10 minutes).
3. Bring the tissue section back to ambient temperature.
4. Wash the tissue section with 0.3 % PBST.
5. Treat the tissue section with 40% methanol containing 0.3 % H₂O₂ (30 minutes)..
6. Block the tissue section with 5% goat serum-PBS (1 hour).
7. Incubate the tissue section with the primary antibody (this product) diluted 100-fold with 0.3 %PBST (4°C, overnight).
8. Wash the tissue section with 0.3 % PBST.
9. Incubate the tissue section with the biotinylated secondary antibody (room temperature, 2 hours).
10. Wash the tissue section with 0.3 % PBST.
11. Incubate the tissue section with DAB. After dehydration treatment, mount the tissue section.

Triton X-100 is a registered trademark of Dow Chemical Company.

Usage example - Flow cytometry