TITLE: Mouse brain hemisphere organotypic cultures on glass coverslips
AUTHORS: Jan Tonnesen

[DESCRIPTION]
This protocol describes preparation of organotypic hemisphere cultures prepared from mouse brain and cultured as rollerdrum cultures on glass coverslips. Having the cultures directly attached to glass coverslips facilitates imaging at very high resolution on inverted microscopes, including time lapse imaging. We specifically use these for STED microscopy based super-resolution microscopy, including super-resolution shadow imaging (SUSHI). 

[STEPS]
SECTION: Preparatory steps
8. Thaw chicken plasma at 4°C, prepare thrombin working solution and keep at 4°C.
SECTION: Preparatory steps
9. Disinfect surgical tools and utensils in pure ethanol and rinse off in dH2O.
SECTION: Preparatory steps
10. Turn on vibratome and set to 250 μm interval slicing.
SECTION: Brain dissections
13. Decapitate mouse pup and submerge the head in ice-cold cutting solution for 30 seconds. Keeping the tissue cold is of utmost importance.
SECTION: Brain dissections
14. 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.
SECTION: Preparatory steps
11. Prepare cutting and dissection solutions, keep on ice. Allow culture medium to warm up (room temperature or 37°C).
SECTION: Brain dissections
15. Cut away cerebellum, and cut brain sagittally into two hemispheres.
SECTION: Slicing on vibratome
16. Glue hemispheres onto slicing platform on caudal surface, so cutting starts rostrally from olfactory bulbs. Embed in low-melt agar for mechanical support.
SECTION: Slicing on vibratome
17. 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.
SECTION: Mounting slices on glass coverslips
18. Collect the slices and place them in ice-cold wash buffer. Discard imperfect slices. 
SECTION: Mounting slices on glass coverslips
19. 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.
SECTION: Mounting slices on glass coverslips
20. Leave at 5 degrees C for 30 min (in fridge).
SECTION: Culturing
21. Place slices on coverslips individually in roller tubes and add 0.75 ml medium to each. Maintain in rollerdrum incubator at 35 degrees C.
SECTION: Culturing
22. Change medium every 5-7 days. Discard cultures that detach, get infected, or do not maintain their gross structure.
SECTION: Culturing
23. Optionally add mitosis inhibitor on day 3-4 and remove after 12-18 hours by changing medium.
SECTION: Preparatory steps
12. Place culture tubes and PLL-coated coverslips at hand.
SECTION: Prepare in advance
1. 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.
SECTION: Prepare in advance
2. 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.
SECTION: Prepare in advance
3. 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)
SECTION: Prepare in advance
4. 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.
SECTION: Prepare in advance
5. 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
SECTION: Prepare in advance
6. 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. 
SECTION: Prepare in advance
7. 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.