Banker Culture Hippocampal Dissection

 

1)       Begin by placing two large (100mm ) and three small (60mm ) petri dishes in an ice bath. Fill with cold HBSS medium (calcium and magnesium free). Place a large Falcon tube (50ml) containing fresh HBSS in bath.

2)       Euthanize the pregnant rat with halothane by placing the animal in a dessicator and applying 2-3ml of halothane (Sigma B-4388) solution to the tissue in the bottom of the container. Ensure that all movements including breathing have ceased. In the flow hood spray the abdomen and instruments with 70% ethanol; change gloves.

3)       To minimize contamination, cut first through the skin and lay it back away from the abdomen. Rinse instruments with 70% ethanol and then cut through the abdomen wall.

4)       Lift out the uterus and sever connections to other tissues. Place in the first large petri dish. Using the lid of a large petri dish and small scissors remove embryos one at a time from the uterus and decapitate them, placing the heads in the second large petri dish.

5)       Clean surface and instruments with ethanol. The heads can be left in the media for between 30 minutes and 1 hour if necessary.

6)       Take one head and using forceps remove the skin covering the skull.

7)       Place forceps under the skull at the base and tear skull using upward movements � peel skull to the side. Using the spatula, scoop the brain out into the first small dish of medium. Repeat for the next 4-5 heads.

8)       Transfer one brain into the second dish of medium and place on the microscope. Turn the brain so that the bottom faces upwards � look for the triangle of blood vessels known as the circle of Willis.

9)       Make two cuts roughly along the area marked out by the blood vessels to separate the hemispheres from the brainstem area and the diencephalon.

10)   The hippocampus is located in the thicker end of the hemisphere. Place forceps in the narrow part of the hemisphere to hold it still and peel away the meninges from the medial part of the hemisphere. Usually it is possible to grasp the meninges and pull it away as a single sheet � but be gentle to avoid tearing the underlying hippocampus.

11)   The inner edge of the hippocampus is free, so only the outer edge and the anterior and posterior ends of the hippocampus have to be cut away from adjoining tissue to remove it from the hemisphere. The best way to make these cuts is to hold the scissors still, and gently steer the hemisphere into the appropriate position for each cut.

12)   Using a glass pipette with a wide opening, or a plastic 1ml pipette, transfer the hippocampus into the third small dish of medium, but try not to deposit much of the medium from the second dish into the third.

Hippocampal Dissociation

 

 

 

 

1)       Transfer the hippocampi to a 15ml conical centrifuge tube containing 3ml HBSS plus 0.5ml of 2.5% trypsin (Gibco 25095-019 HBSS) and tap the tube to mix. Unlike glia, no DNase is added to the neuronal dissociation medium. Place tube in incubator for 15 minutes.

 

 

2)       After the incubation the hippocampi will be at the bottom of the tube and the medium can be aspirated off. Be careful to leave 0.5ml solution at the bottom of the tube because the cells are very sticky � if one gets sucked off the rest are likely to follow. Add 13ml of medium to the hippocampi, shake the tube to mix and let stand for 5 minutes. Remove solution. Repeat twice more to allow residual trypsin to diffuse from the tissue.

3)       Bring the final volume to 5ml and pipette gently up and down. This step should be performed 10 times with a slightly narrowed siliconized pasteur pipette and then 10 times with a pipette whose tip has been fire polished to half the normal diameter (do this by heating the pipette tip in a bunsen flame while slowly turning the pipette). To prevent foaming eject the HBSS out of the pipette against the side of the centrifuge tube.

4)       Pipette until the solution looks slightly cloudy with few dense particles of tissue.

 

 

 

Plating Neurons

 

 

1)       Place a few µl of solution under the coverslip on a haemocytometer. (Smith lab use a 0.1mm deep Reichert).

 

 

2)       In a large square (5 x 5 small squares) it should be possible to count approximately 40-60 cells.

 

 

3)       If there are 40 cells, plate 1ml of suspension into a 100mm petri dish containing 10ml neuronal medium + 10% horse serum. There should be enough suspension to plate 4 x 100mm petri dishes, each containing around 6 x 25mm coverslips. If there are 60 cells per large square, reduce the amount of suspension added to the petri dish (i.e. add 650 µl).

 

 

4)       After pipetting cells into the petri dish, swirl the solution around gently so that the cells will spread to all coverslips in the dish. Place the dish in the incubator.

 

 

5)       After 3-4 hours the coverslips should be flipped cell-side down onto the dish of glia.

 

 

 

 

 

Before starting the dissociation , make sure the 100mm dishes containing coverslips and neuronal medium, plus the glial dishes with neuronal medium, are ready in the incubator.

 

 

<center> <p>  </p> </center>
上一篇：Cryotomy 详细介绍冷冻切片的方法   下一篇：Quick and simple mouse forebrain commissure anatomy