Sep 14, 2024
Isolation of the Region Around Locus Coeruleus for Single Nucleus RNA Profiling
- Pauline Jakobs1,
- Diana Municchi1,
- Matthias Prigge1
- 1Leibniz Institute for Neurobiology
- TeamPrigge
Protocol Citation: Pauline Jakobs, Diana Municchi, Matthias Prigge 2024. Isolation of the Region Around Locus Coeruleus for Single Nucleus RNA Profiling. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxy4x4l8j/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 26, 2024
Last Modified: September 15, 2024
Protocol Integer ID: 106432
Keywords: ASAPCRN
Funders Acknowledgement:
ASAP and MJFF
Grant ID: ASAP-020505
Disclaimer
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Abstract
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.
Tissue Isolation of the Region Around Locus Coeruleus for Single Nucleus RNA Profiling
After punching peri-LC region, you can also shock freez in liquid nitrogen-
Buffer Preparation (important - steril filter and autoclave)
Buffer Preparation (important - steril filter and autoclave)
Prepare the buffer a day before, and keep it in the fridge before using
Stock solution to acuratly prepare working solution!
~ 500ml Milli-Q-Water
add reagents of Table 1
fill up until 1000 ml
sterile filter
Bubble with 95% O2 and 5% CO2 until ph is stable (approx. 15-20 min)
adjust ph with HCl -> must have: 7,3-7,4 pH, 315 mOsm Can be stored at 4°C for a couple of days.
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
Preparation for Brain Extraction
Preparation for Brain Extraction
- 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
Clean EVERYTHING with RNase free!
Prepare FALCON 3.1 Falcon with ACSF and FBS (0,5%)
250 µl FBS + 50 ml aCSF 🡪 close with Parafilm to prevent gas leakage 3.2 Falcon with ACSF and Protease (1mg/ml)
30 mg Protease + 30 ml ACSF
Prepare chambers for recovery of brain slices
Chamber with NMDG-aCSF (RT): Recovery of Slices
Chamber with RT NMDG-aCSF + Protease
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
Place Instruments on a towel for dissection and trituration → clean with RNase free
- Prepare Vibratome: new blade each time!
80 Hz, 200 Frequency, Section: 300 µm (LEICA VT1200)
Procedure of extracting brain and cutting brain stem slices
Procedure of extracting brain and cutting brain stem slices
Euthanize mice with 0.1 ml pento or other approved euthanizing method
Decapitate, remove skin, open scull (window)
Remove brain with spatula Block Brain (depending on region of interest (ROI)
Glue agarose block to platform Place and glue brain in front of agarose block
Place platform in vibratome, add ice cold NMDG-ACSF, bubble with 5 % CO2 und 95 % O2
Vibratome: 80 Hz, Amplitude 200, section 300 µm, duration 0,10 mm/s.
Discard brain slices until Locus coeruleus is reached
Place brain slices into ice-cold NMDG-ACSF bubble (5 % CO2 und 95 % O2) for 15 min Recovery
Extracting and Isolating peri-LC region
Extracting and Isolating peri-LC region
- Fill Petri dish with NMDG-aCSF+FBS
Place slices with pasteur pipette into petri dish
When working with fluorescence labeled cells turn off light!
Punch out brain areas with biopsy puncher (1.5 mm)
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
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)
If you immediately proceed with nuclei isolation, follow the protocol provided by the 10X Nuclei Isolation Kit (link)
Place samples on ice until further usage