Jul 10, 2024
Multicolor fluorescence in situ hybridization and analysis
- Chuyu Chen1
- 1Northwestern University, Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Protocol Citation: Chuyu Chen 2024. Multicolor fluorescence in situ hybridization and analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4937rgo5/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: July 10, 2024
Last Modified: July 10, 2024
Protocol Integer ID: 103165
Keywords: ASAPCRN, molecule fluorescence in situ hybridization, lrrk2 kinase activity, multicolor fluorescence in situ hybridization, impact of lrrk2 kinase activity, molecule fluorescence, signaling pathway, kinase activity, pathways in ispn, multicolor fluorescence, fluorescence, expression of several immediate early gene, several immediate early gene, d2r antagonism, iegs in the ispn
Funders Acknowledgements:
Aligning Science Across Parkinson's [ASAP-020600] through the Michael J. Fox Foundation for Parkinson's Research (MJFF)
Grant ID: ASAP-020600
Abstract
Antipsychotics are known to induce the expression of several immediate early genes (IEGs) via D2R antagonism, which then leads to changes in critical signaling pathways in iSPNs. To define the impact of LRRK2 kinase activity on the expression pattern of IEGs in the iSPNs, we used multicolored single-molecule fluorescence in situ hybridization.
Fresh Frozen Tissue Prep
Place a piece of aluminum foil into a small Styrofoam container containing dry ice.
Mice were euthanized with carbon dioxide, decapitated, their brains rapidly removed, and placed immediately on foil on dry ice
Pour ~50 mL of 2-methylbutance into the container. Keep brain in container for 2 minutes.
2m
When the brain is completely frozen, carefully remove it from the foil with forceps and place it in
the OCT mold.
Pour ~50 mL of 2-methylbutance into the container. Keep brain in container for 3 minutes.
3m
Cover the top of the mold with tin foil and store the brain in -80°C freezer. Place the brain into -20°C freezer 20 mins before cryosection.
collect the region of interest with 20μm slices and mount by turning the slide upside down and pressing against the slice on theplatform.
Place slides in slide box and store slides in -80°C freezer.
Fix and Dehydrate slides
Prepare 200 mL of 4% PFA and chilled to 4°C:
Place slides in the staining tank containing chilled 4% PFA in fridge (4°C) for 30mins
30m
Wash slides in 1X PBS by moving the rack gently up and down for 2 minutes and repeat with fresh 1X PBS (room temperature)
2m
Place slide rack in new staining tank containing 200 mL of 50% EtOH for 5 minutes at room temperature. Gently agitate slides by moving rack up and down 2-4 times
5m
Place slide rack in new staining tank containing 200 mL of 70% EtOH for 5 minutes at room temperature. Gently agitate slides by moving rack up and down 2-4 times.
5m
Turn on the HbyEZ oven and prepare the humidity contral tray by applying ~40 mL of MilliQ water to paper towels.
Place slide rack in new staining tank containing 200 mL of 100% EtOH for 5 minutes at room temperature. Gently agitate slides by moving rack up and down 2-4 times.
5m
Allow Protease IV to come to RT (normally stored at 4°C)
Place slide rack in new staining tank containing 200 mL of 100% EtOH for 5 minutes at room temperature. Gently agitate slides by moving rack up and down 2-4 times.
5m
Warm probes for 10-15 minutes at 40°C
15m
Practice making hydrophobic barriers using ImmEdge pen
Remove probes from oven, allow to come to RT
Pretreat samples with Hydrogen Peroxide and Protease
48m
Load the slides into the EZ-Batch holder, and add 5 drops of RNAscope Hydrogen Peroxide to each section (completely cover the sections). incubate at RT for 10 minutes.
10m
Place the EZ-Batch holder into the wash tray containing 200ml distilled water.
2m
Repeat the wash step with fresh water.
2m
Add 5 drops of RNAscope Protease IV to each section (completely cover the sections). incubate at RT for 30 minutes.
30m
Place the EZ-Batch holder into the wash tray containing 200ml PBS
2m
Repeat the wash step with fresh PBS
2m
Allow slides to dry. Collect excess PBS with Kimwipe
Multicolor fluorescence in situ hybridization
2h
Preparing probes by ratio 50:1:1 for channels 1, 2, and 3
Add mixed probes to each section. Apply ~100 μL of probe combination to each section (completely cover the sections) and incubate at 40°C for 2hrs.
2h
30 minutes before the end of incubation: remove amps from the fridge and allow amps to come to RT
30m
Flick off excess liquid and wash slides in 1X Wash Buffer (200ml) for 2 minutes
2m
Repeat with fresh 1X Wash Buffer in fresh wash buffer
2m
Remove excess liquid
Apply ~3 drops of Amp 1 to each section(completely cover the sections) and incubate at 40°C for 30 minutes.
30m
Flick off excess liquid and wash slides in 1X Wash Buffer (200ml) for 2 minutes
2m
Repeat with fresh 1X Wash Buffer in fresh wash buffer
2m
Remove excess liquid
Apply ~3 drops of Amp 2 to each section(completely cover the sections) and incubate at 40°C for 30 minutes
30m
Flick off excess liquid and wash slides in 1X Wash Buffer (200ml) for 2 minutes
2m
Repeat with fresh 1X Wash Buffer in fresh wash buffer
2m
Remove excess liquid
Apply ~3 drops of Amp 3 to each section(completely cover the sections) and incubate at 40°C for 15 minutes
15m
Flick off excess liquid and wash slides in 1X Wash Buffer (200ml) for 2 minutes
2m
Repeat with fresh 1X Wash Buffer in fresh wash buffer
2m
Remove excess liquid
Apply ~3 drops of HRP-C1 to each section(completely cover the sections) and incubate at 40°C for 15 minutes
15m
Flick off excess liquid and wash slides in 1X Wash Buffer (200ml) for 2 minutes
2m
Repeat with fresh 1X Wash Buffer in fresh wash buffer
2m
Remove excess liquid
Apply 150μl of Opal 520 to each section(completely cover the sections) and incubate at 40°C for 30 minutes
30m
Flick off excess liquid and wash slides in 1X Wash Buffer (200ml) for 2 minutes
2m
Repeat with fresh 1X Wash Buffer in fresh wash buffer
2m
Remove excess liquid
Apply ~3 drops of HRP blocker to each section(completely cover the sections) and incubate at 40°C for 15 minutes
15m
Flick off excess liquid and wash slides in 1X Wash Buffer (200ml) for 2 minutes
2m
Repeat with fresh 1X Wash Buffer in fresh wash buffer
2m
Remove excess liquid
Apply ~3 drops of HRP-C2 to each section(completely cover the sections) and incubate at 40°C for 15 minutes
15m
Flick off excess liquid and wash slides in 1X Wash Buffer (200ml) for 2 minutes
2m
Repeat with fresh 1X Wash Buffer in fresh wash buffer
2m
Remove excess liquid
Apply 150μl of Opal 570 to each section(completely cover the sections) and incubate at 40°C for 30 minutes
30m
Flick off excess liquid and wash slides in 1X Wash Buffer (200ml) for 2 minutes
2m
Repeat with fresh 1X Wash Buffer in fresh wash buffer
2m
Remove excess liquid
Apply ~3 drops of HRP blocker to each section(completely cover the sections) and incubate at 40°C for 15 minutes
15m
Flick off excess liquid and wash slides in 1X Wash Buffer (200ml) for 2 minutes
2m
Repeat with fresh 1X Wash Buffer in fresh wash buffer
2m
Remove excess liquid
Apply ~3 drops of HRP-C3 to each section(completely cover the sections) and incubate at 40°C for 15 minutes
15m
Flick off excess liquid and wash slides in 1X Wash Buffer (200ml) for 2 minutes
2m
Repeat with fresh 1X Wash Buffer in fresh wash buffer
2m
Remove excess liquid
Apply 150μl of Opal 650 to each section(completely cover the sections) and incubate at 40°C for 30 minutes
30m
Flick off excess liquid and wash slides in 1X Wash Buffer (200ml) for 2 minutes
2m
Repeat with fresh 1X Wash Buffer in fresh wash buffer
2m
Remove excess liquid
Apply ~3 drops of HRP blocker to each section(completely cover the sections) and incubate at 40°C for 15 minutes
15m
Flick off excess liquid and wash slides in 1X Wash Buffer (200ml) for 2 minutes
2m
Repeat with fresh 1X Wash Buffer in fresh wash buffer
2m
Remove excess liquid
Apply ~3 drops of DAPI to each section(completely cover the sections) and incubate at RT for 1 minutes
1m
Flick off excess liquid and mount the slides with Prolong Diamond Antifade mountant.
Allow slides to dry in protected and covered area overnight. store slides in the dark at 4°C
Confocal images capture
Sections were imaged with the Nikon A1 laser microscope system using a 60X 0.75NA objective, to capture 3 z-stack images across 4 channels:
Image analysis with Cell Profiler
DAPI channel was enhanced with Enhance Or Suppress Features module, with feature size set at pixel size 30.
Identify Primary Objects module was implemented on identified nuclei, object pixel unit was set for Min 5, Max 50, with threshold strategy set as Global and thresholding method set as Otsu
Drd1 or Drd2 channel was enhanced with Enhance Or Suppress Features module, with feature size set at pixel size 10
Nr4a1 or Arc channel was enhanced with Enhance Or Suppress Features module, with feature size set at pixel size 5.
Identify Primary Objects module was implemented on identified Drd1 or Drd2 signal puncta, object pixel unit was set for Min 3, Max30, threshold strategy for Global and thresholding method set as Manual
Nur77 or Arc object pixel unit was set for Min 1, Max10, threshold strategy for Global and thresholding method set as Manual
Masked D1 positive nuclei and Masked D2 positive nuclei were generated with RelateObjects
The number of objects were measured with the Measure Object Intensity module.