I. Viral injection in organotypic slice culture

• Virus type : Sindbis virus

• Injection time : DIV 4-5 days in case of slices made with p7~8 rat pups

• Incubation after injection : 18-24 hrs in 35 degree incubator

• Specificity : Change culture media after injection.

Procedure

1. Put culture media 2 ml to a dish (Felcon 353002) and warm up in the incubator.

2. Preparation of glass pipettes.

- Using micropuller (Shutter Ins. P-97) and glass electrodes (WPI, Item No. 4878), pull micro pipettes (SCJ Ver. Diameter ~2 um after pulling with Program No. 19 of puller).

- Using the micro forge (Narishige MF-830), break the tip of micro pipettes to make the tip size 15~20 um (SCJ Ver. No heat use, Just push down the micro pipette to the stiff wire held in the micro forge).

- Prepare the 1 ml syringe and fill it with “mineral oil (Sigma M5904)”. Set the MicroFil (WPI MF34G-5) on the tip of syringe instead of a syringe needle.

- Autoclave glass pipettes (tip broken) and the syringe (with oil and MicroFil) under the UV (Kodak EDAS290) for 15 min.

3. Set up the microinjector.

- Clean up the table with 70% alcohol tissue.

- Set up the Microscope (Zeiss StemiSV6) and microinjector (WPI Nanoliter 2000) on the table, and clean up them with 70% alcohol tissue.

4. Prepare the virus.

- Prepare ice in the small bawl.

- Take out the virus from deep freezer and keep it cold in ice.

- Wait until virus is neutrally melted.

5. Injection to slices.

- Take out the micropipette and the syringe from UV box.

- Fill the micropipette up with mineral oil by using the prepared syringe.

- Set the micropipette (filled with mineral oil) in the microinjector (Nanolitter 2000)

Check three gaskets (two black and one white)

See the manual how to set up gaskets in the micropipette

- Empty the micropiptte up to the end (using the injector controller)

-Set the Parafilm under microscope and drop 5~10 ul virus on the middle of film.

-Locate the tip of micropipette on the drop of virus, seeing through the microscope.

- Fill the micropipette with virus by using the injector controller

- Take out the warmed dish (see procedure 1) and slice plates from incubator and place them in the clean bench.

- Move one culture membrane (with slices) from the plate to the dish.

- Locate the dish with slices under the microscope.

- Locate the tip of micropipette on the proper area of the slice (CA1 area including soma of pyramidal neurons. SCJ Ver. Just touch the surface of slices and don’t push the micropipette deeply. It can damage slices)

- Shot once on the located area by using controller and move the micropipette gently to the second position (SCJ Ver. The second position is generally selected 1 - 2 mm away from the first injected area)

- Shot once on the second area

- Repeat same procedure with other slices.

- After injecting all slices of the plate, change the culture media and return it back to the incubator.

- Incubation for 18-24 hrs before recording.

Tips (SCJ version) 

1. Kv4.2 and Kv4.2W362F have different properties from each other. One and two days after injection, Kv4.2-infected neurons are best to be recorded. However, use Kv4.2W362F-infjected neurons only one day after injections. Longer incubation can make neurons so weak in case of Kv4.2W362F.

2. Other constructs (Kchip, DPPX etc) may have different infection properties, so discuss with Dr. Hoffman and lab members before using them.

3. You should keep all items and slices for injection from the contaminating conditions. If slices are contaminated, you should find another work for a week.

4. Don’t use bad slices for infections. Bad slices do not show any CA3, CA1 and DG lines clearly. In this case, it is difficult to find CA1 neurons and infected neurons would be dead even if you could infect them.

5. Virus for the organotypic slice injection should have higher titers than dissociated neurons. Virus condition should be better for organotypic slices. Sometimes, Virus, which is working well in dissociated neurons, might not be working in the organotypic slices.

6. Well-infected and health neurons should show a long green dendrite under the microscope.