Sep 16, 2024
Tissue preparation and tissue imaging for MERFISH
- Cameron Oram1
- 1McGill University
Protocol Citation: Cameron Oram 2024. Tissue preparation and tissue imaging for MERFISH. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn6opyl5d/v1
Manuscript citation:
Molecular and spatial transcriptomic classification of midbrain dopamine neurons and their alterations in a LRRK2G2019S model of Parkinson’s disease
Zachary Gaertner, Cameron Oram, Amanda Schneeweis, Elan Schonfeld, Cyril Bolduc, Chuyu Chen, Daniel Dombeck, Loukia Parisiadou, Jean Francois Poulin, Rajeshwar Awatramani
bioRxiv 2024.06.06.597807; doi: https://doi.org/10.1101/2024.06.06.597807
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: September 16, 2024
Last Modified: September 17, 2024
Protocol Integer ID: 107703
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020600
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
MERSCOPE Sample preparation protocol and imaging protocol
Materials
General Reagents:
● 32% paraformaldehyde (PFA) – (Electron Microscopy Sciences, 15714S)
● 10x PBS, DNAse, RNAse free - (Thermofisher, AM9625)
● Sucrose - (Sigma Aldrich, S9378)
● Ethanol (EtOH) – (Sigma, E7023)
● DNAse, RNAse free water
● Tissue-Tek OCT compound (VWR, 4583)
● RNAse inhibitor, murine – (NEB, M0314S)
MERSCOPE Reagents:
- MERSCOPE Sample Prep Kit (Vizgen, 10400012) containing:
- o Sample Prep Buffer
- o Formamide Wash Buffer
- o Sample prep clearing premix
- o Gel embedding premix
- Custom 500 gene panel premix
- MERSCOPE Slide Box (Vizgen, 10500001)
Perfusion and sectioning of brain
Perfusion and sectioning of brain
C57Bl6 male mice were housed in groups until 2-3 months of age.
4% PFA solution was made from 32% PFA concentrate in 1x PBS.
Mice were perfused first with 25 mL of chilled 1x PBS (DNAse, RNAse free) followed by 50 mL of chilled 4% PFA. Brains were extracted and post-fixed in PFA for 16:00:00 .
16h
Brains were transferred to 30% Sucrose until fully submerged.
Brains were then submerged in OCT solution and frozen on dry ice.
10um coronal sections of ventral midbrain were obtained with a cryostat and placed onto specialized MERSCOPE slides.
MERSCOPE sample preparation
MERSCOPE sample preparation
Each step of the following protocol was performed in RNAse free conditions using RNAse away and RNAse free solutions.
Each brain slice to be imaged was first placed into a 60mm petri dish and washed 3 times in 5 mL 1x PBS for 00:05:00 .
5m
Add 5 mL of 70% EtOH, cover the petri dish with parafilm and incubate at 4 °C for 16:00:00 to permeabilize the tissue.
16h
Aspirate the EtOH and wash 1 time with 5 mL Sample Prep Buffer.
Aspirate the sample prep buffer and incubate with5 mL of Formamide Wash Buffer at 37 °C for 00:30:00 .
30m
Aspirate as much of the formamide wash buffer from the dish and slide without disturbing the tissue. Then add 50 µL of the custom MERSCOPE gene panel mix. Place a small piece (2cm x 2cm) of fresh parafilm on top of the tissue section without disturbing it or introducing air bubbles.
Clean the outside of the petri dish with 70% EtOH and place into a tissue culture incubator (humidified, 37 °C ) for 36-48 hours
Remove the parafilm and add 5 mL of formamide wash buffer. Incubate at 47 °C for 00:30:00 .
30m
Aspirate the wash buffer and add another 5mL formamide was buffer to the petri dish. Continue incubating at 47 °C for another 00:30:00 .
30m
Wash 1 time with 5 mL sample prep buffer for 00:05:00 .
5m
Prepare Gel Coverslips by spraying with RNAse away and cleaning with a kimwipe. Then wash with 70% EtOH and clean again with a kimwipe followed by lens cleaning paper. Add 100 µL of Gel Slick solution to the coverslip and allow to evaporate at room temperature (approximately 10 minutes).
Prepare the gel embedding solution by adding 5mL Gel embedding premix to 25uL of 10% Ammonium persulfate solution and 2.5 µL of TEMED. Retain 100 µL of the embedding solution into a separate Eppendorf.
Aspirate the sample prep buffer and add the remaining 5 mL of gel embedding solution to the petri dish. Incubate at room temperature for 00:01:00 .
1m
Aspirate the gel embedding solution and add 50 µL of the retained solution directly on top of the tissue section. With forceps, place the cleaned glass coverslip on top of the tissue (gel slick coated side facing the tissue). Allow the gel embedding solution to spread across the entire coverslip and aspirate any excess solution. Incubate at room temperature for 01:30:00 to allow the gel to solidify.
1h 30m
Carefully remove the coverslip without disrupting the tissue.
Warm the clearing solution at 37 °C for 00:30:00 prior to use. For each sample, prepare 5 mL of clearing solution by adding 50uL of Proteinase K. Add the clearing solution to the petri dish and incubate at 47 °C for 24:00:00 .
1d 0h 30m
Once the tissue has fully cleared, wash the petri dish with sample prep buffer (approximately 3 times, 5 mL for 00:05:00 on rotation). Add DAPI and PolyT solution to the petri dish and incubate at room temperature for 10 minutes. Wash extensively with sample prep buffer and prepare the sample for imaging.
5m
Imaging
Imaging
1h
1h
Imaging cartridges are first thawed in 37 °C water bath for 01:00:00 before imaging. After a complete thaw is finished, puncture a hole in the fluid container and add 100 µL of RNAse inhibitor, pipetting up and down 10 times to mix the contents.
1h
Slides are loaded into the slide holding contraption and connected to the Merscope input and output tubing. The imaging cartridge is inserted to the device and imaging fluid is pulled through the slide holder.
4x images of the brain tissue is collected and the area of interest is circled with the Vizgen imaging software. The device is allowed to run over the course of 24-48 hours until all regions of interest are imaged.