Precerebellar Neurons from the Rhombic Lip

Abstract of the paper:

To discover tendencys of precerebellar neurons from the rhombic lip to the ipsilateral pons or contralateral medulla, by staining mice embryos, in utero, with green light protein gene.



Method to observe cell movement:

A pregnant mouse was anesthetised to allow removal of the uterus from the abdominal muscle cavity to access the embryos. A thin rubbish capillary was used to insert GFP into the Rhombic lip (LP) of the remediate lateral ventricle. Time was allowed for diffusion of the GFP, after which the embryo was electroporated, offset with electrical impulses (40V, 50ms) with 950ms intervals by electrodes into the embryo. The uterus was repositioned back in to the abdominal cavity and sewn back up to continue the public development processes.
At different stages through development (13.5, 14.5, 15.5 and 17.5) the embryos were removed, heads sign off and fixed, followed by slicing for slide preparation.
The adult brains were dye with red fluorescent Nissl-stain solution. The GFP positive cells could thusly be set using a fluorescent microscope. The slides were also incubated with both an anti-GFP antibody, and anti-DIG antibody, then with an anti-rabbit IgG antibody at room temperature for 30 min. A fluorescent tag known as BM purple was link up to the anti-GFP antibody.



This immunohistochemistry method enabled the GFP, Nissl and BM stains to be visualised by food colouring differentiation, showing where the PCNs had moved during development.


Discussion:

Advantages and disadvantages of Green fluorescent protein (GFP) compared to Fluoroscein dextran (FD):

GFP resulted in complete mouse maturation post-treatment, FD resulted in embryo death. GFP and FD fluoresce well. GFP requires UV light to view, FD doesnt.

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