Aug 23, 2024

Public workspaceRNAscope for FFPE Mouse Tissue

DOI
dx.doi.org/10.17504/protocols.io.81wgbzwkqgpk/v1
  • 1University of Minnesota
  • Team Lee
Jane Balster
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DOI: dx.doi.org/10.17504/protocols.io.81wgbzwkqgpk/v1
Protocol Citation: Hector Martell Martinez 2024. RNAscope for FFPE Mouse Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbzwkqgpk/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 21, 2024
Last Modified: August 23, 2024
Protocol Integer ID: 106357
Keywords: ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: 000592
Abstract
This protocol details the RNAscope for FFPE Mouse Tissue.
Materials
RNAscope Multiplex Fluorescent Reagent Kit v2 (Cat. #323100).ReagentRNAscope® Multiplex Fluorescent Reagent Kit v2Advanced Cell DiagnosticsCatalog #323100

Day 1:
  • Fresh Xylene
  • Absolute alcohol
  • Hydrogen peroxide & Protease plus from same kit
  • Absorbent paper
  • 10X Target retrieval
  • C1, C2, C3 probes
  • ReagentRNAscope™ 3-plex Negative Control ProbeAdvanced Cell DiagnosticsCatalog #320871
  • ReagentRNAscope™ 3-plex Positive Control Probe- MmAdvanced Cell DiagnosticsCatalog #320881
  • 1X wash buffer
Day 2:
  • AMP1, AMP2, AMP3
  • HRP-C# (need C1-C3 if developing all 3 channels)
  • TSA buffer
  • HRP-Blocker
  • ReagentOpal 570 Reagent PackPerkin ElmerCatalog #FP1488001KT
  • ReagentOpal 690 Reagent PackPerkin ElmerCatalog #FP1497001KT
  • TSA-DIG & Opal 780 (Cat. #FP1501001KT)
  • 1X Antibody Diluent (Cat. #ARD1001EA)
  • DAPI
  • ReagentTrueBlack® Plus Lipofuscin Autofluorescence Quencher, 40X in DMSOBiotiumCatalog #23014
  • ReagentProLong™ Gold Antifade MountantInvitrogen - Thermo FisherCatalog #P36930
  • Cover Slip
  • Tween 20
  • PBS

Solutions

1X Wash Buffer
  • Amount40 mL 50X WashBuffer + Amount1960 mL RNase-free water
Target Retrieval
  • Amount25 mL 10X Target Retrieval + Amount225 mL RNase-free water
5x SSC
  • Amount50 mL 20X SSC + Amount150 mL RNase-freewater
TSA-DIG: for 8 samples
  • Amount2 µL TSA-DIG + Amount1000 µL TSA buffer
Opal 780 Dye: for 8 samples
  • Amount8 µL opal 780 + Amount1000 µL 1X Antibody Diluent
1X TrueBlack Plus: for 8 samples
  • Amount25 µL 40X TrueBlack Plus + Amount1000 µL 1X PBS → VORTEX




DAY 1 - Bake/Adhere
DAY 1 - Bake/Adhere
1h
1h
Spray RNase away on bench top, slide holder, metal container.
Warm oven and slide holder up to Temperature60 °C .

Temperature
Label the lower part of the slide with a pencil.

  • Can place slides on bench (since sprayed with RNase away).
Put slides into slide holder and place into oven → bake for Duration01:00:00 @ Temperature60 °C .

  • Meanwhile..
1h
Temperature
Turn plate warmer on to Temperature60 °C .
Temperature
Make Target Retrieval solution.
Fill Xylene and Ethanol containers.

Note
After Baking: Possible stopping point: Store @ TemperatureRoom temperature , good for ~Duration168:00:00 .

Temperature
Set hyb oven temp to Temperature40 °C .

Temperature
Wet humidifying paper with nanopure water (does not need to be dripping).
Place paper on bottom of slide tray and put into the oven for Duration00:30:00 @ Temperature40 °C .

30m
Temperature
Place slides into tissue tek container and transport to fume hood.
DAY 1 - Deparaffinize
DAY 1 - Deparaffinize
5m
5m


Xylene container 1 for Duration00:05:00 .

5m
Meanwhile..Turn on vegetable steamer.
When transferring to container 2 → shake off excess.
Xylene container 1 for Duration00:05:00 .
5m
When transferring to ethanol → shake off excess.

Absolute ethanol container 1 for Duration00:02:00 .
2m
When transferring to container 2 → shake off excess.
Absolute ethanol container 2 for Duration00:02:00 .
2m
Dry on slide warmer for ~Duration00:05:00 @ Temperature60 °C until dry.

5m
Temperature
Meanwhile... clean bench with RNase away.
DAY 1 - Hydrogen Peroxide
DAY 1 - Hydrogen Peroxide
10m
10m
Place slides on bench top and cover tissue completely with Hydrogen Peroxide (~5-8 drops).

Incubate for Duration00:10:00 @ TemperatureRoom temperature .

  • Meanwhile..

  1. Boil Target Retrieval solution and a container of Nano pure water in microwave until boiling.

10m
Incubation
Temperature
*Microwave for Duration00:01:00 at a time*
1m
When reaches a boil → place containers in warm steamer to ensure solution is at least Temperature99 °C .

Temperature
Remove solution using absorbent paper by tapping long side on paper.

  • Place in slide holder.
Wash slides in slide holder with Amount200 mL of Nano pure water ~3-5 dunks.

Wash
Take slides out and repeat step Go to -Go to 1x.

DAY 1 - Target Retrieval Step
DAY 1 - Target Retrieval Step
10s
10s
Keep in water and transport to steamer.
Dunk a couple times and soak for Duration00:00:10 in nanopure water (steamer).

10s
Place in target retrieval solution in the steamer for Duration00:30:00 (for brain samples) or Duration00:15:00 (for other tissue).

45m
Rinse slides in fresh Amount200 mL of Nano pure water → Duration00:00:15 .

15s
Wash
Transfer slides to the 2nd container of absolute ethanol in the fume hood → Duration00:03:00 .

3m
Dry slides in slide warmer - ~Duration00:05:00 @ Temperature60 °C .

  • Meanwhile..
5m
Temperature
Rinse slide holder in DI water in sink and let dry on paper towel.
Wash
Spray bench with RNase away.
DAY 1 - Barrier/Protease Plus
DAY 1 - Barrier/Protease Plus
30m
30m
Put dry slides on bench and square off with hydrophobic pen.
Leave a little extra room at one side of square to allow space to aspirate later.
Apply ~5 drops of protease plus to tissue until sample is completely covered.
Incubate in oven for Duration00:30:00 @ Temperature40 °C .

  • Meanwhile..
30m
Incubation
Temperature
Warm probes @ Temperature40 °C for ~Duration00:10:00 .

10m
Temperature
Create probe solution.

Note
C1 probe is at 1X → assign to low expresser gene
C2 & C3 probes are at 50 X → dilute w/ C1 probe if using C1 or with probe diluent to 1X

Wash with DI water in wash tray - 2X.
Wash
Aspirate with Amount200 µL pipette tip in the fume hood.
Pipetting
Wipe bottom of wash tray with big kimwipe.
DAY 1 - Hybridize Probe
DAY 1 - Hybridize Probe
30m
30m
Add ~Amount120 µL of probe mix to respective samples → cover sample completely.
Pipetting
Incubate for Duration02:00:00 @ Temperature40 °C .

2h
Incubation
Temperature
Wash in 1X wash buffer for 2 minutes 2X.

Note
Store till day 2 in 5X SSC @ TemperatureRoom temperature .

  • Pour ethanol back into reagent bottle if not doing RNAscope within the next week so doesn't evaporate.
Wash
Temperature
Wash in 1X wash buffer for Duration00:02:00 (1/2).
2m
Wash
Wash in 1X wash buffer for Duration00:02:00 (2/2).
2m
Wash
DAY 2
DAY 2
2m
2m
Spray RNase away on bench top, slide holder, metal container.
Hydrate Paper and turn on oven to Temperature40 °C .

Temperature
Wash slides with 1X wash buffer for 2 minutes with slight agitation 2X.

Wash
Wash slides with 1X wash buffer for Duration00:02:00 with slight agitation (1/2).
2m
Wash
Wash slides with 1X wash buffer for Duration00:02:00 with slight agitation (2/2).
2m
Wash
Aspirate.
Hybridize AMP1.
4-5 drops covering the sample with AMP1.
Incubate for Duration00:30:00 @ Temperature40 °C .

30m
Incubation
Temperature
Wash slides with 1X wash buffer for 2 minutes with slight agitation 2X.

  1. Wash slides with 1X wash buffer for Duration00:02:00 with slight agitation (1/2).
  2. Wash slides with 1X wash buffer for Duration00:02:00 with slight agitation (2/2).

4m
Wash
Aspirate.
Hybridize AMP2.
4-5 drops covering the ample with AMP2.
Incubate for Duration00:30:00 @ Temperature40 °C .
30m
Incubation
Temperature
Wash slides with 1X wash buffer for 2 minutes with slight agitation 2X.

  1. Wash slides with 1X wash buffer for Duration00:02:00 with slight agitation (1/2).
  2. Wash slides with 1X wash buffer for Duration00:02:00 with slight agitation (2/2).

4m
Wash
Aspirate.
Hybridize AMP3.
4-5 drops covering the sample with AMP3.
Incubate for Duration00:15:00 @ Temperature40 °C .
15m
Incubation
Temperature
Wash slides with 1X wash buffer for 2 minutes with slight agitation 2X.

  1. Wash slides with 1X wash buffer for Duration00:02:00 with slight agitation (1/2).
  2. Wash slides with 1X wash buffer for Duration00:02:00 with slight agitation (2/2).

4m
Wash
Aspirate.
Develop Channels (C1-C3)
Develop Channels (C1-C3)
1h 6m
1h 6m

Note
  • Don’t need to do all 3 channels for each sample just for respective channels, do one channel at a time/slide.
  • PC and NC need all 3 Channels.
  • Develop 780 channel Last!!
Develop HRP-C2 (570 or 690) signal.
Add 4-5 drops covering the sample with HRP-C2 (or respective HRP-C#) and incubate for Duration00:15:00 @ Temperature40 °C .

  • Meanwhile.. Dilute Opal dyes → KEEP IN DARK
Amount1 µL Dye (570 or 690) + Amount1000 µL TSA buffer

15m
Incubation
Temperature
Wash slides with 1X wash buffer for 2 minutes with slight agitation 2X.

  1. Wash slides with 1X wash buffer for Duration00:02:00 with slight agitation (1/2).
  2. Wash slides with 1X wash buffer for Duration00:02:00 with slight agitation (2/2).

  • Aspirate.
4m
Wash
Add 1st Opal dye and Incubate for Duration00:30:00 @ Temperature40 °C .
30m
Incubation
Temperature
Wash slides with 1X wash buffer for 2 minutes with slight agitation 2X.

  • Wash slides with 1X wash buffer for Duration00:02:00 with slight agitation (1/2).
  • Wash slides with 1X wash buffer for Duration00:02:00 with slight agitation (2/2).

  • Aspirate.
4m
Wash
Add 4-6 drops HRP blocker and incubate for Duration00:15:00 @ Temperature40 °C .
15m
Incubation
Temperature
Wash slides with 1X wash buffer for 2 minutes with slight agitation 2X.

  1. Wash slides with 1X wash buffer for Duration00:02:00 with slight agitation (1/2).
  2. Wash slides with 1X wash buffer for Duration00:02:00 with slight agitation (2/2).

  • Aspirate.
4m
Wash
Repeat Go to using HRP-C3.

For 780 Channel
Add HRP-C1 and incubate for Duration00:15:00 @ Temperature40 °C .

  • Wash slides with 1X wash buffer for 2 minutes with slight agitation 2X.

  1. Wash slides with 1X wash buffer for Duration00:02:00 with slight agitation (1/2).
  2. Wash slides with 1X wash buffer for Duration00:02:00 with slight agitation (2/2).

  • Aspirate.
19m
Incubation
Wash
Temperature
Add ~Amount120 µL of TSA-DIG per section and incubate for Duration00:30:00 @ TemperatureRoom temperature .

  • Wash slides with 1X wash buffer for 2 minutes with slight agitation 2X.

  1. Wash slides with 1X wash buffer for Duration00:02:00 with slight agitation (1/2).
  2. Wash slides with 1X wash buffer for Duration00:02:00 with slight agitation (2/2).

  • Aspirate.
34m
Incubation
Wash
Temperature
Add HRP Blocker and incubate for Duration00:15:00 @ Temperature40 °C .

  • Wash slides with 1X wash buffer for 2 minutes with slight agitation 2X.

  1. Wash slides with 1X wash buffer for Duration00:02:00 with slight agitation (1/2).
  2. Wash slides with 1X wash buffer for Duration00:02:00 with slight agitation (2/2).

  • Aspirate.
19m
Incubation
Wash
Temperature
Add ~Amount120 µL of Opal 780 per section and incubate for Duration00:30:00 @ TemperatureRoom temperature .

  • Wash slides with 1X wash buffer for 2 minutes with slight agitation 2X.

  1. Wash slides with 1X wash buffer for Duration00:02:00 with slight agitation (1/2).
  2. Wash slides with 1X wash buffer for Duration00:02:00 with slight agitation (2/2).

  • Aspirate.
34m
Incubation
Wash
Temperature
Quench w/ TrueBlack Plus
Quench w/ TrueBlack Plus
10m
10m
Wash w/ PBS 3X.

  • Aspirate.
Wash
Apply ~Amount120 µL of 1X TrueBlack Plus for Duration00:10:00 @ TemperatureRoom temperature .

10m
Temperature
Wash w/ PBS 3X.

  • Aspirate.
Wash
DAPI and Mount
DAPI and Mount
30s
30s
Add 4-5 drops of DAPI to completely cover section.
Incubate for Duration00:00:30 @ TemperatureRoom temperature .

30s
Incubation
Temperature
Remove DAPI with absorbent paper → tap long side gently to paper.
Add 3-4 drops of Prolong Gold Mountant.
Add Coverslip.
Use a forceps to attach coverslip.
Gently press down.
Kim wipe the bottom of the slide and the bench in between samples.
Store in dark DurationOvernight on absorbent paper (in a drawer).

  • Dry slides in the dark overnight and then Store slides at Temperature4 °C the next day.

30s
Overnight
Temperature
Protocol references
Refer to ACD RNAscope Multiplex Fluorescent v2 Assay for reference (doc #323100).