Anti Mouse IgG, Goat, SPICA DyeTM 647 Conjugated

Anti Mouse IgG, Goat, SPICA Dye™ 647 Conjugated is a secondary antibody conjugated with SPICA Dye™, a novel ultra-photostable fluorescent dye developed by Fujifilm. Compared to conventional fluorescent dyes, SPICA Dye™ is significantly more resistant to photobleaching. This antibody is suitable for both immunohistochemistry and immunocytochemistry.

Antibody Information

Clonality	Polyclonal
Antigen	Mouse IgG
Host	Goat
Formulation	PBS, 0.05% Sodium azide
Conjugate	SPICA Dye™ 647
Cross-reactivity	Mouse
Application	Immunohistochemistry (frozen section) 1:500-1,000 Immunocytochemistry 1:500-1,000

Excitation/Emission

SPICA Dye™ 647

(Excitation=651 nm, Emission=670 nm)

Protocol (Example)

Immunohistochemistry of Mouse Brain Sections using Anti-GFAP Antibody and Anti Mouse IgG, Goat, SPICA Dye™ 647 Conjugated

1.Perfusion fixation

Perfuse with 0.1% 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 °C, from overnight to 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% BSA and 2% normal donkey serum in 0.3% Triton X-100/PBS (RT, 2 hours)

6.Primary antibody reaction

Anti GFAP, Monoclonal Antibody(MO389) (Product Number 018-27283) 1:1,000（4 °C, Overnight)

7.Washing

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

8.Secondary antibody reaction

Anti Mouse IgG, Goat, SPICA Dye™ 647 Conjugated (This product) 1:500 in 0.3% Triton X-100/PBS (RT, 2 hours)

9.Washing

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

10.Mounting

The sample is sealed in mounting medium (ex. Invitrogen™ ProLong Glass) and stored at 4 °C in a dark place.

Performance Data

Photostability Evaluation Using Xenon Light Resistance Test Compared to Conventional Dyes

Using a xenon light resistance tester equipped with a 500 W xenon lamp and a glass filter, PBS solutions containing goat anti-rabbit IgG antibodies conjugated with either SPICA Dye™ 647 or Alexa Fluor^® 647 (Jackson ImmunoResearch Inc., Product Number 111-005-003) were continuously subjected to xenon light irradiation. Changes in absorbance at the maximum absorption wavelength were plotted to compare the photostability of the two dyes.

[Result]
The SPICA Dye™ 647-conjugated antibody demonstrated greater photostability than the antibody conjugated with the competitor’s fluorescent dye.

Photostability Comparison in Immunohistochemistry

Immunostained mouse cryosections were subjected to 10 rounds of Z-stack acquisition using a confocal microscope, and fluorescence intensity was measured.

Species	Mouse (C57BL6/J)
Site	Hippocampus
Sample	Frozen section
Primary Antibody	Anti GFAP, Monoclonal Antibody(MO389) (Product Number 018-27283) 1:500
Secondary Antibody	Anti Mouse IgG, Goat, SPICA Dye™ 647 Conjugated (This product) 1:500 Alexa Fluor® 647-AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson ImmunoResearch Inc., Product Number 115-605-146) 1:500

Condition

Microscope	CLSM (Nikon, ECLIPSE Ti)
Objective lens	Plan Apo VC ×20 (NA 0.75)
Excitation	647 nm
Detection	660-1,000 nm
Steps	3.0 μm x 8 Steps
Scan Speed	0.125 Frame/sec (pixel Dwell/5.3 μsec)

[Result]
The SPICA Dye™ 647-conjugated antibody exhibited slower photobleaching than the Alexa Fluor^® 647-conjugated antibody.

* Alexa Fluor^® is a registered trademark of Invitrogen Corporation.