Mouse brain hemisphere organotypic cultures on glass coverslips

Cutting solution:

Sucrose 195 mM

KCl 2.5 mM

NaH₂PO₄ 1.25 mM

NaHCO₃ 28 mM

CaCl₂ 0.5 mM

L-ascorbic acid 1 mM,

pyruvic acid 3 mM

glucose 7 mM

MgCl₂ 7 mM

Should be continuously equilibrated with 5% CO2 in oxygen (carbogen bubbling) during use.

Washing solution :

97 ml HBSS

1 ml Kynurenic acid (of 0.2M stock to reach 2 mM)

0.46 ml Glucose (11.5 mM, from 2.5M stock)

1 ml HEPES (of 2M stock to reach 20mM)

1 ml Pen/Strep (1x)

Adjust pH with 1M HCL or NaOH to 7.2, sterile filter and store at 4°C up to two days.

Culturing medium:

50 ml MEM-Medium (with Hank’s salts)

20 ml HBSS

20 ml horse serum

1 ml Glutamax

2 ml B27

0.46 ml Glucose (11.5 mM final)

1 ml Sucrose (20mM final)

0.5 ml HEPES (10 mM final)

0.5 ml Pen/Strep (0.5x recommended)

3.5 mg Ascorbic acid (0.2mM final)

PLL-coated glass coverslips:

Wash/sterilize coverslips (by ethanol wash and or UV light) and place a drop of 0.01% Poly-L-lysine solution on the center part covering some 50% of the total area. Leave for 1-4 hours. Wash 3 times with dH₂O and allow to dry. Can be stored in fridge or freezer for at least 2 weeks.

Thrombin stock and working solution:

Stock solution: 0,5 g Thrombin in 50 ml dH₂O+ 50 ml HBSS solution and store in 500 μl aliquots at -20°C.

Work solution:

742,5 μl HBSS

4 μl Glucose from 2.5M stock (final 11.5 mM)

500 μl Thrombin stock

Chicken plasma solution:

Add 5 ml dH₂O per bottle using injection needle, briefly vortex, wait 5 min, sterile filter and store in 500 μl aliquots at -20°C until use.

Mitosis inhibitor:

12.1 mg of Uridine in 50 ml dH₂O

13.9 mg of Ara-C in 50 ml dH₂O

12.3 mg of 5-Fluoro-2’-deoxyuridin in 50 ml dH₂O

Mix 10ml of each solution, sterile filter and store in 500μl aliquots at -20°C for 3-6 months .

Use at 10 μl per culturing tube.

Thaw chicken plasma at 4°C, prepare thrombin working solution and keep at 4°C.

Disinfect surgical tools and utensils in pure ethanol and rinse off in dH₂O.

Turn on vibratome and set to 250 μm interval slicing.

Prepare cutting and dissection solutions, keep on ice. Allow culture medium to warm up (room temperature or 37°C).

Place culture tubes and PLL-coated coverslips at hand.

Decapitate mouse pup and submerge the head in ice-cold cutting solution for 30 seconds. Keeping the tissue cold is of utmost importance.

Cut skin on scalp along midline starting caudally. Cut skull similarly and bent out skull to sides, to expose brain. Spoon out brain into ice-cold cutting solution using spatula.

The dissection should be swift and take no longer than 1 min.

Cut away cerebellum, and cut brain sagittally into two hemispheres.

Glue hemispheres onto slicing platform on caudal surface, so cutting starts rostrally from olfactory bulbs. Embed in low-melt agar for mechanical support.

Mount slice platform with hemispheres in vibratome and embed in ice-cold cutting solution. Start slicing at 250 μm interval until the region of interest is reached. Collect slices consecutively and maintain in room temperature cutting solution.

Collect the slices and place them in ice-cold wash buffer. Discard imperfect slices.

Using inverted Pasteur glass pipette, collect individual slices and place on PLL-coated coverslips. Remove excess medium, and add first 10 μl of chicken plasma solution, and then 10 μl of thrombin solution. Mix well around and embed slice.

Leave at 5 degrees C for 30 min (in fridge).

Place slices on coverslips individually in roller tubes and add 0.75 ml medium to each. Maintain in rollerdrum incubator at 35 degrees C.

Change medium every 5-7 days. Discard cultures that detach, get infected, or do not maintain their gross structure.

Optionally add mitosis inhibitor on day 3-4 and remove after 12-18 hours by changing medium.