Imaging Hippocampal Neuronal Activation In Mouse Brain Sections

Samenvatting

Source: Provenzano, G., et al. Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders. J. Vis. Exp. (2015)

This video demonstrates immunohistochemical imaging to visualize hippocampal neuronal activation. Stained brain sections reveal darker regions under a bright-field microscope, indicating activated neurons.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Immunohistochemistry

1. Transfer 3-5 dorsal hippocampal sections for each mouse into 24 multi-well plates containing 500 µl of 0.1 M phosphate-buffered saline (PBS) for each well.
2. Wash the sections contained in each well with 500 µl of 0.1 M PBS (5 min, 3 times with ....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
24 well plate	Sigma	CLS3524
100% ethanol	Fisher Scientific	A406-20	Used to make ethanol gradient for dehydration prior to slide mounting.
Xylene	VWR	66004-950	Toxic - to be used under hood. Change xylene every month depending on use.
PBS	Sigma	P3813-10PAK
Triton X-100	Sigma	T-8787
Hydrogen peroxide	Sigma	H1009-100ML
Normal goat serum	Abcam	G9023-10ML
ABC kit Vectastain	Vector Laboratories	PK-6100	Add in a volume of 5 ml of PBS 2 drops of reagent A, mix and then add 2 drops of reagent B and mix.
DAB peroxidase substrate	Vector Laboratories	SK-4100	Add in a volume of 5 ml double distilled water: 2 drops of buffer stock solution and mix; 4 drops of DAB and mix; 2 drops of hydrogen peroxide and mix.
pERK antibody	Cell Signaling Technologies	4370	Dilution 1:500
Biotinylated goat anti-rabbit IgG antibody	Vector Laboratories	BA-1000	Dilution 1:250
SuperFrost Slides	Carl Roth	1879
Coverslips	Fisher	12-548-B
DPX	Sigma	317616	Mounting medium for slides. Equivalent mounting medium can be used.