TITLE: Isolation of the Region Around Locus Coeruleus for Single Nucleus RNA Profiling
AUTHORS: Pauline Jakobs, Diana Municchi, Matthias Prigge

[DESCRIPTION]
This protocol outlines the methodology for isolating the peri-locus coeruleus (peri-LC) region in mice for subsequent single nucleus RNA profiling. Key steps include the preparation of NMDG-aCSF buffer with precise pH and osmolarity, careful euthanasia and extraction of the brain, and the use of a vibratome to obtain 300 µm thick slices. The peri-LC region is identified and isolated using a biopsy puncher, followed by immediate shock-freezing in liquid nitrogen to preserve tissue integrity. This approach facilitates high-quality RNA profiling, essential for understanding the molecular characteristics of the locus coeruleus and its surrounding regions.

[STEPS]
SECTION: Buffer Preparation (important - steril filter and autoclave)
3. Prepare the buffer a day before, and keep it in the fridge before using
SECTION: Buffer Preparation (important - steril filter and autoclave)
5. ~ 500ml Milli-Q-Water
SECTION: Buffer Preparation (important - steril filter and autoclave)
6. add reagents of Table 1
SECTION: Buffer Preparation (important - steril filter and autoclave)
7. fill up until 1000 ml 
SECTION: Buffer Preparation (important - steril filter and autoclave)
8. sterile filter 
SECTION: Buffer Preparation (important - steril filter and autoclave)
9. Bubble with 95% O2 and 5% CO2  until ph is stable (approx. 15-20 min) 
SECTION: Buffer Preparation (important - steril filter and autoclave)
10. adjust ph with HCl -> must have: 7,3-7,4 pH, 315 mOsm  Can be stored at 4°C for a couple of days.
SECTION: Buffer Preparation (important - steril filter and autoclave)
11. On the day of the experiment add freshly: + 10,0 g Trehalose+ 50 µl (0,1 µM) TTX+ 1 ml (200 µM) DNQX+ 1 ml (50 µM) APV+ 1 µg/ml actinomycin-d 
SECTION: Preparation for Brain Extraction
12. Prepare NMDG-aCSF
sterile filtered pH 7,3 
🡪 Important between 7,3 and 7,4!! 300 mOsm after Trehalose 
+ 10 mg/ 1L Trehalose 
Stir while bubbling with 5 % CO2 and 95 % O2. 
for 15- 20 Min and check pH 7,3

SECTION: Preparation for Brain Extraction
13. Clean EVERYTHING with RNase free!
SECTION: Preparation for Brain Extraction
14. Prepare FALCON 3.1 Falcon with ACSF and FBS (0,5%)

SECTION: Preparation for Brain Extraction
15. Prepare chambers for recovery of brain slices 
SECTION: Extracting and Isolating peri-LC region
29. When working with fluorescence labeled cells turn off light!
SECTION: Extracting and Isolating peri-LC region
30. Punch out brain areas with biopsy puncher (1.5 mm)
SECTION: Preparation for Brain Extraction
14.1. 250 µl FBS + 50 ml aCSF 🡪 close with Parafilm to prevent gas leakage 3.2 Falcon with ACSF and Protease (1mg/ml) 
SECTION: Preparation for Brain Extraction
14.2. 30 mg Protease + 30 ml ACSF
SECTION: Preparation for Brain Extraction
17. Place Instruments on a towel for dissection and trituration → clean with RNase free
SECTION: Preparation for Brain Extraction
18. Prepare Vibratome: new blade each time! 
80 Hz, 200 Frequency, Section: 300 µm (LEICA VT1200)
SECTION: Extracting and Isolating peri-LC region
28. Fill Petri dish with NMDG-aCSF+FBS
 Place slices with pasteur pipette into petri dish
SECTION: Preparation for Brain Extraction
16. Prepare Agaroseblock to assist vibrotome slicing and preventing pushing brain away during slicing
2% Agarose in Petri dish (fridge). 
Cut out small block approx. 1,5 cm – 1,5 cm

SECTION: Preparation for Brain Extraction
15.1. Chamber with NMDG-aCSF (RT): Recovery of Slices 
SECTION: Preparation for Brain Extraction
15.2. Chamber with RT NMDG-aCSF + Protease
SECTION: Procedure of extracting brain and cutting brain stem slices
20.  Euthanize mice with 0.1 ml pento or other approved euthanizing method
SECTION: Procedure of extracting brain and cutting brain stem slices
21. Decapitate, remove skin, open scull (window)
SECTION: Procedure of extracting brain and cutting brain stem slices
22. Remove brain with spatula Block Brain (depending on region of interest (ROI) 
SECTION: Procedure of extracting brain and cutting brain stem slices
23. Glue agarose block to platform Place and glue brain in front of agarose block  
SECTION: Procedure of extracting brain and cutting brain stem slices
24. Place platform in vibratome, add ice cold NMDG-ACSF, bubble with 5 % CO2 und 95 % O2
SECTION: Procedure of extracting brain and cutting brain stem slices
25. Vibratome: 80 Hz, Amplitude 200, section 300 µm, duration 0,10 mm/s.
SECTION: Procedure of extracting brain and cutting brain stem slices
26. Discard brain slices until Locus coeruleus is reached 
SECTION: Procedure of extracting brain and cutting brain stem slices
27. Place brain slices into ice-cold NMDG-ACSF bubble (5 % CO2 und 95 % O2) for 15 min Recovery
SECTION: Extracting and Isolating peri-LC region
33. If you immediately proceed with nuclei isolation, follow the protocol provided by the 10X Nuclei Isolation Kit (link)
SECTION: Extracting and Isolating peri-LC region
34. Place samples on ice until further usage
SECTION: Preparation for Brain Extraction
19. 
 

SECTION: Extracting and Isolating peri-LC region
31. In this image the LC is accumulating NM and iseasily visible. Landmarks such as the 4th vetricle can help you to locate LC and peri-LC region
 

SECTION: Extracting and Isolating peri-LC region
32. After punching the peri-LC region, store the tissue in a small PCR tube and immediately shock-freeze it in liquid nitrogen. Ensure the tissue is sent to collaborators as intact tissue, not as isolated nuclei.


 
A biopsy tissue puncher with diameter of 1.5mm is recommended. We used biopuncher from Miltex (image copywrite Miltek/Tedpella  https://www.tedpella.com/histo_html/miltex-plunger-punch.aspx)
1. Tissue Isolation of the Region Around Locus Coeruleus for Single Nucleus RNA Profiling
2. 
 

SECTION: Buffer Preparation (important - steril filter and autoclave)
4.