Mar 10, 2025

Public workspaceMultiplex fluorescence in situ hybridization using RNAScope on paraffin embedded human nervous system tissue with optional photobleaching for autofluorescence mitigation

DOI
dx.doi.org/10.17504/protocols.io.j8nlk81wxl5r/v1
  • 1National Institutes of Health, Clinical Center;
  • 2University of Pennsylvania;
  • 3National Institutes of Health, NINDS;
  • 4National Institutes of Health, NICHD;
  • 5AnaBios Corporation;
  • 6National Institutes of Health, NIA, NIAAA
Matthew R. Sapio
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DOI: dx.doi.org/10.17504/protocols.io.j8nlk81wxl5r/v1
Protocol Citation: Matthew R. Sapio, Diana King, Dragan Maric, Samay R. Shah, Thomas Talbott, Allison P. Manalo, Pranavi Nara, Wenting Ma, Andre Ghetti, Christopher E. Ramsden, Michael Iadarola, Andrew J Mannes 2025. Multiplex fluorescence in situ hybridization using RNAScope on paraffin embedded human nervous system tissue with optional photobleaching for autofluorescence mitigation. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk81wxl5r/v1
Manuscript citation:
Sapio MR, King DM, Maric D, Shah SR, Talbot TL, Manalo AP, Nara P, Ma W, Ghetti A, Ramsden CE, Iadarola MJ, Mannes AJ. Efficient removal of naturally-occurring lipofuscin autofluorescence in human nervous tissue using high-intensity white light. J Pain. 2025 Mar 6:105359. doi: 10.1016/j.jpain.2025.105359. Epub ahead of print. PMID: 40057214.

https://pubmed.ncbi.nlm.nih.gov/40057214/
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 29, 2024
Last Modified: March 10, 2025
Protocol Integer ID: 106701
Keywords: lipofuscin, autofluorescence, multiplex fluorescence , microscopy, central nervous system, molecular neuroscience, anatomy
Funders Acknowledgements:
Intramural Research Program of the National Institutes of Health, Clinical Center
Grant ID: 1ZIACL090033, 1ZIACL090034, and 1ZIACL090035
Intramural Research Program of NCCIH
Grant ID: 1ZIAAT000017-03
Intramural Research Program of NINDS
Grant ID: 1ZICNS009441-02
Abstract
This protocol is to perform the RNAScope in situ hybridization procedure in human central nervous system tissue with modifications from our newly established method to reduce endogenous autofluorescence from lipofuscin. This procedure should increase signal to noise by drastically reducing fluorescence background during microscopic image capture.
Image Attribution
Image of human somatosensory cortical neurons after photobleaching to remove endogenous autofluorescence (Figure 5). In situ hybridization targets: SLC17A7 (Green), LIMK1 (Yellow), GAD1 (Orange), STX1A (Magenta).
Materials
Equipment
Air cooled Peltier cold plate (ThermoElectric Cooling America Corporation.)
High intensity LED light source with manifold. Described in associated manuscript.
HybEZ‱ II Hybridization System for Manual Assays (Advanced Cell Diagnostics)

Kit reagents from Advanced Cell Diagnostics
RNAscope Multiplex Fluorescent Kit v2 (Advanced Cell Diagnostics)
RNAscope 4-plex Ancillary Kit for Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostics)

Fluorophores from Akoya Biosciences
Opal‱ 520 Reagent Pack (Akoya Biosciences)
Opal‱ 570 Reagent Pack (Akoya Biosciences)
Opal‱ 620 Reagent Pack (Akoya Biosciences)
Opal‱ 690 Reagent Pack (Akoya Biosciences)

Other reagents
Epredia‱ Immu-Mount‱ (Fisher Scientific)
ImmEdge® Pen (Vector, Cat no. H-4000)

Chemical reagents
Xylenes
95% ethanol
Optional exposure to high-intensity light to reduce endogenous autofluorescence in human nervous system tissues
Optional exposure to high-intensity light to reduce endogenous autofluorescence in human nervous system tissues
Set cold plate to 2°C.
Place slides inside a clean clear plastic bag to prevent condensation from forming on top of the slides. Use plastic spacers (such as caps of 50 ml tubes) to prevent contact between bag and slides.


Note
For formalin fixed paraffin embedded (FFPE) slides, do not deparaffinize at this stage. For optimal results for visualization of mRNAs, cut sections should be stored at -80°C until use.

Incubate slides for 24-72 hours under light exposure.
Day 1 of RNAScope™ procedures
Day 1 of RNAScope™ procedures
1h
1h
Bake paraffin-embedded slides in oven at Temperature60 °C for Duration01:00:00

1h
During baking, prepare staining bins (for dipping microscopy slides). 2 staining bins will be needed filled with fresh xylenes, and 2 with fresh 95% ethanol.
Safety information
Xylenes should be handled in a chemical fume hood.

Toxic
Place slides in plastic slide holder and perform the following rinses in a chemical fume hood. Agitate briefly:
Wash in xylenes (x2) Duration00:05:00
Wash in 95% ethanol (x2) Duration00:02:00

7m
During the xylene wash steps above, set a hot plate to Temperature100 °C . Heat Amount200 mL of 1x target retrieval solution. Cover with foil to insulate, and maintain the temperature at Temperature100 °C ±Temperature2 °C by monitoring with a temperature probe. Heat at this temperature for Duration00:15:00 .
15m
Let ethanol evaporate off of the slides (dry completely).
Add H202 in drops to cover tissue sections, and let sit for Duration00:10:00 at TemperatureRoom temperature .

10m
During H202 application step, begin preparing probes.
a. Warm probes at Temperature40 °C for Duration00:10:00 , then cool to TemperatureRoom temperature .
b. Spin Channel 2-4 probes (C2-C4) down to collect liquid at the bottom of the tube.
c. Add necessary volumes of probes to one tube - total volume varies as needed, with approximately 50:1:1:1 ratio of Channel 1 (C1) probe to C2-C4. (ex. add Amount1 µL of C2, C3 and C4 probes to Amount47 µL C1 probe.)
d. Label probe mixes and gently agitate to mix.

10m
Prepare 2 staining bins of distilled H20 (dH20).
Tap off H202 (stored at Temperature4 °C ) from slides, and rinse briefly in dH20 (x2).

Place sections in pre-prepared Temperature100 °C 1x target retrieval buffer (step 3.1) for Duration00:25:00 .
Note
Target retrieval step can vary by tissue type. The general guideline is 15-25 minutes.



25m
Prepare staining bin of room temperature dH20, and staining bin of 95% ethanol.
Rinse sections in dH20 for Duration00:00:15 , then rinse in 95% ethanol for Duration00:03:00 . Let slides dry completely after rinsing in ethanol.

3m 15s
Draw on hydrophobic barrier around sections. Allow to dry for Duration00:05:00 .
Note
We recommend Vector ImmEdge® Pen (Cat no. H-4000). The choice of pen in this step is critical for desired results.




5m
Critical
Prepare slide tray for oven with blotting/filter paper on the bottom (soaked in dH20) to keep slides from drying out during the subsequent heating steps.
Add protease plus reagent to slides dropwise to cover samples. Place in oven to bake at Temperature40 °C for Duration00:30:00 .
Note
Protease reagent type, and time of this digest can vary by tissue type. If subsequent immunohistochemistry is desired, a less stringent protein digest may be beneficial.


30m
Prepare a staining bin of dH20.
Tap off protease reagent, and wash slides in dH20 briefly with agitation.
Tap off dH20 and cover samples with probe mixture. Return slides to oven at Temperature40 °C for Duration02:00:00 to hybridize probes to tissue.

2h
Prepare 2 staining bins of 1x wash buffer. To prepare the wash buffer, warm the 50x stock to Temperature40 °C and dilute in dH20.

Wash slides in 1x wash buffer twice for Duration00:02:00 with initial agitation.

2m
Fill staining bin with 5x SSC buffer (from 50x stock) and store samples overnight for day 2.
Day 2 of RNAScope™ procedures
Day 2 of RNAScope™ procedures
30m
30m
Set out kit reagents for Amp1-3 and HRP-C1, -C2, -C3, -C4 and HRP blocker and allow to come to room temperature.
Wash slides for ~2 min in 1x wash buffer.
Prepare slide tray for oven with blotting/filter paper on bottom soaked with dH20 to keep slides from dying out while in oven.
The following steps are repetitive wash and incubation steps. This checklist is useful to track progress over time.
Checklist for day 2 protocol procedures.
The following steps are repetitive wash and incubation steps. This checklist is useful to track progress over time.

*Row 5 is an additional set of steps performed for four-plex only. Individuals performing three-plex can stop after completing Row 4.


Line 1 of above chart:
Tap off liquid and add Amp1 to cover tissue within hydrophobic barrier.
Incubate at Temperature40 °C for Duration00:30:00 .
Tap off Amp1 and wash slides for ~2 min in 1x wash buffer (twice).
30m
Tap off liquid and add Amp2 to cover tissue within hydrophobic barrier.
Incubate at Temperature40 °C for Duration00:30:00 .
Tap off Amp2 and wash slides for ~2 min in 1x wash buffer (twice).

30m
Tap off liquid and add Amp3 to cover tissue within hydrophobic barrier.
Incubate at Temperature40 °C for Duration00:15:00 .
Tap off Amp3 and wash slides for ~2 min in 1x wash buffer (twice).
15m
Line 2 of above chart:
Tap off liquid and add HRP-C1 to cover tissue within hydrophobic barrier.
Incubate at Temperature40 °C for Duration00:15:00 .
Tap off HRP-C1 and wash slides for ~2 min in 1x wash buffer (twice).
15m
Tap off liquid and add 150-200 µl diluted fluorophore for C1 to each slide.
Incubate at Temperature40 °C for Duration00:30:00 .
Tap off fluorophore and wash slides for ~2 min in 1x wash buffer (twice).
30m
Tap off liquid and add HRP blocker to cover tissue.
Incubate at Temperature40 °C for Duration00:15:00 .
Tap off HRP blocker and wash slides for ~2 min in 1x wash buffer (twice).
15m
Line 3 of above chart:
Tap off liquid and add HRP-C2 to cover tissue within hydrophobic barrier.
Incubate at Temperature40 °C for Duration00:15:00 .
Tap off HRP-C2 and wash slides for ~2 min in 1x wash buffer (twice).
15m
Tap off liquid and add 150-200 µl diluted fluorophore for C2 to each slide.
Incubate at Temperature40 °C for Duration00:30:00 .
Tap off fluorophore and wash slides for ~2 min in 1x wash buffer (twice).
30m
Tap off liquid and add HRP blocker to cover tissue.
Incubate at Temperature40 °C for Duration00:15:00 .
Tap off HRP blocker and wash slides for ~2 min in 1x wash buffer (twice).
15m
Line 4 of above chart:
Tap off liquid and add HRP-C3 to cover tissue within hydrophobic barrier.
Incubate at Temperature40 °C for Duration00:15:00 .
Tap off HRP-C3 and wash slides for ~2 min in 1x wash buffer (twice).
15m
Tap off liquid and add 150-200 µl diluted fluorophore for C3 to each slide.
Incubate at Temperature40 °C for Duration00:30:00 .
Tap off fluorophore and wash slides for ~2 min in 1x wash buffer (twice).
30m
Tap off liquid and add HRP blocker to cover tissue.
Incubate at Temperature40 °C for Duration00:15:00 .
Tap off HRP blocker and wash slides for ~2 min in 1x wash buffer (twice).
15m
Line 5 of above chart:
Tap off liquid and add HRP-C4 to cover tissue within hydrophobic barrier.
Incubate at Temperature40 °C for Duration00:15:00 .
Tap off HRP-C4 and wash slides for ~2 min in 1x wash buffer (twice).
15m
Tap off liquid and add 150-200 µl diluted fluorophore for C4 to each slide.
Incubate at Temperature40 °C for Duration00:30:00 .
Tap off fluorophore and wash slides for ~2 min in 1x wash buffer (twice).
30m
Tap off liquid and add HRP blocker to cover tissue.
Incubate at Temperature40 °C for Duration00:15:00 .
Tap off HRP blocker and wash slides for ~2 min in 1x wash buffer (twice).
15m
Add ~4 drops of DAPI solution to each slide and incubate for ~30 seconds at room temperature, then immediately tap off.

Coverslip using Immu-Mount and store 30 min to overnight in the dark. Longer term storage should be done at 4°C.

Note
Immu-Mount is useful because it is reversible if multiple rounds of staining are desired.

Protocol references
1               Goto, T. et al. Longitudinal peripheral tissue RNA-Seq transcriptomic profiling, hyperalgesia, and wound healing in the rat plantar surgical incision model. FASEB J 35, e21852 (2021). https://doi.org/10.1096/fj.202100347R
2               Goto, T. et al. Longitudinal Transcriptomic Profiling in Carrageenan-Induced Rat Hind Paw Peripheral Inflammation and Hyperalgesia Reveals Progressive Recruitment of Innate Immune System Components. J Pain 22, 322-343 (2021). https://doi.org/10.1016/j.jpain.2020.11.001
3               Kim, J. J. et al. Transcriptional Activation, Deactivation and Rebound Patterns in Cortex, Hippocampus and Amygdala in Response to Ketamine Infusion in Rats. Front Mol Neurosci 15, 892345 (2022). https://doi.org/10.3389/fnmol.2022.892345
4               Sapio, M. R. et al. Comparative Analysis of Dorsal Root, Nodose and Sympathetic Ganglia for the Development of New Analgesics. Front Neurosci 14, 615362 (2020). https://doi.org/10.3389/fnins.2020.615362
5               Sapio, M. R. et al. Expression pattern analysis and characterization of the hereditary sensory and autonomic neuropathy 2 A (HSAN2A) gene with no lysine kinase (WNK1) in human dorsal root ganglion. Exp Neurol 370, 114552 (2023). https://doi.org/10.1016/j.expneurol.2023.114552
6               Sapio, M. R. et al. Analgesic candidate adenosine A3 receptors are expressed by perineuronal peripheral macrophages in human dorsal root ganglion and spinal cord microglia. Pain (2024). https://doi.org/10.1097/j.pain.0000000000003242
7               Ma, W. et al. Anatomical Analysis of Transient Potential Vanilloid Receptor 1 (Trpv1+) and Mu-Opioid Receptor (Oprm1+) Co-expression in Rat Dorsal Root Ganglion Neurons. Front Mol Neurosci 15, 926596 (2022). https://doi.org/10.3389/fnmol.2022.926596