Application
Immunohistochemistry free floating
Sample
Mouse Tissue sections (Hippocampus CA1 mouse Brain)
Specification
Hippocampus CA1 mouse Brain
Other product details
Incubation time
24 hour(s) and 0 minute(s) · Temperature: 4°C
Dilution
1/500
Secondary antibody
Name
Non-Abcam antibody was used: Goat anti Rabbit A488 from Thermo Fisher Scientifi
Host species: Goat
Clonality: Polyclonal
Conjugation: Alexa Fluor® 488
Host species: Goat
Clonality: Polyclonal
Conjugation: Alexa Fluor® 488
Additional data
Additional Notes
Paraformaldehyde fixation: Mice were anesthetized and perfused intracardially with 1X PBS, followed by a 4% paraformaldehyde solution 10 min. To ensure complete tissue fixation, the brains were removed and transferred into 4% paraformaldehyde overnights at 4°C and then stored into 20-30% sucrose in preparation to sectioning (Brains were allowed to completely
sink to the bottom of the container before sectioning). Brains were cut sagittally into 25 µm slices using a cryostat and slices stored in a cryo-protectant solution at -20°C.
Immunofluorescence staining protocol: Free flooding sections were washed with 1X PBS then pre-incubated with acetone 7-10 min. Then washed 5 times with 1X PBS and incubated with a blocking buffer (10% normal goat serum NGS/ in 1X TBS containing 0.3% Triton X-100 for 1 hr). Sections were then incubated overnight at 4oC on an orbital rotator with primary antibodies in 3% bovine serum albumins (BSA) in 1X PBS containing 0.1% Tween 20. The day after, following five times washing with 1X PBS buffer solution, sections were incubated with appropriate Alexa secondary antibodies at a 1:250 dilutions in 3% BSA/PBST, followed by 5 washing with 1X PBS buffer solution. Prior to mounting on glass microscope slides, slices were rinsed with water.
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投稿 Jan 19 2016