Sep 20, 2023
Dual In Situ Hybridization/Immunofluorescence
- Michael Henderson1
- 1Van Andel Institute
Protocol Citation: Michael Henderson 2023. Dual In Situ Hybridization/Immunofluorescence. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l61n91vqe/v1
Manuscript citation:
Adapted from ACD Standard Protocol/Cheadle/Otero-Garcia Protocols
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: January 05, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 56572
Keywords: Dual In Situ Hybridization, Immunofluorescence, RNAscope Multiplex Fluorescent v2 Assay, ASAPCRN
Abstract
This protocol details about the Dual In Situ Hybridization/Immunofluorescence in tissue.
Attachments
338-741.pdf
527KB
Guidelines
ACD protocol notes that tissues should be fixed in 10% NBF for 16-32 hours, and embedded in paraffin. Then, sectioned and dried overnight at RT. They suggest sectioned tissue be used in less than a year (4°C) or less than 3 months at room temperature.
Materials
Solutions
| A | B | C | |
| Needed (mL) | Stock Solution | Final Concentration | |
| 5 L | dH2O | ||
| 485 g | Tris base | 0.5 M | |
| 240 mL | Concentrated HCl | ||
| pH to 7.6 | |||
| To 8L | dH2O |
0.5 M Tris (8 L)
Reagents
| A | B | C | D | E | |
| Vendor | Catalog # | Qty | Unit Price | Description | |
| RNAscope® Multiplex Fluorescent Reagent Kit V2 | 323100 | 1330 | Contains H2O2, protease reagents, target retrieval reagent, wash buffer, HRP reagents | ||
| RNAscope® 3-plex | 320861 | 100 | Polr2a (C1 channel) and PPIB (C2 channel), UBC (C3 channel) | ||
| Positive Control Human Sigma | 199664-25G | 1 | 66.6 | Sudan Black B | |
| Vector Laboratories | H-4000 | 1 | 120 | ImmEdge Hydrophobic Barrier Pen | |
| Southern Biotech | 0100-01 | 1 | 45.14 | DAPI Fluoromount-G |
Sudan black BSigma AldrichCatalog #199664
ImmEdge hydrophobic barrier pap penVector LaboratoriesCatalog #H-4000
Fluoromount-GSouthern BiotechCatalog #0100-01
Preparing Tissue (Day 1): Prepare Tissue
Preparing Tissue (Day 1): Prepare Tissue
1h 19m
1h 19m
Bake slides in a dry oven for 01:00:00 at 60 °C . Use slides within a week.
1h
De-paraffinize slides in fresh xylenes, then in 100% ethanol.
De-paraffinize slides for 00:05:00 in fresh xylenes. (1/4)
5m
De-paraffinize slides for 00:05:00 in fresh xylenes. (2/4)
5m
De-paraffinize slides for 00:02:00 in 100% ethanol. (3/4)
2m
De-paraffinize slides for 00:02:00 in 100% ethanol. (4/4)
2m
Place slides on absorbent paper and dry in the oven from00:05:00 at 60 °C or until dry.
5m
Preparing Tissue (Day 1): Hydrogen Peroxide Treatment
Preparing Tissue (Day 1): Hydrogen Peroxide Treatment
1h 19m
1h 19m
Place slide horizontally in an incubation tray. Add ~5-8 drops of RNAscope Hydrogen Peroxide to cover each section. Incubate for 00:10:00 at Room temperature .
10m
Dab solution off and move to a rack in distilled water. Move up and down 5 times. Repeat with a fresh boat of distilled water.
Preparing Tissue (Day 1): Target Retrieval
Preparing Tissue (Day 1): Target Retrieval
1h 19m
1h 19m
Dilute Target Retrieval Regents (RNAscope) 1:10 in dH2O (25 mL /225 mL dH2O/boat). Mix well.
Place in microwave for 00:15:00 at 95 °C .
15m
Transfer slides to a slide boat with 200 mL distilled water for 00:00:15 .
15s
Transfer the slides to 100% ethanol for 00:03:00 .
3m
Dry the slides in a 60 °C incubator (or Room temperature ) for 00:05:00 .
5m
Draw a hydrophobic barrier onto slides with ImmEdge pen. Do NOT due for fluorescent slides. Let the barrier dry for00:05:00 . OPTIONAL PAUSE POINT Overnight atRoom temperature .
10m
RNAscope Multiplex Fluorescent v2 Assay (Day 2): Protease Treatment
RNAscope Multiplex Fluorescent v2 Assay (Day 2): Protease Treatment
1h 15m
1h 15m
Place a wet Humidifying Paper in an incubation tray and warm for 00:30:00 at 40 °C (TC incubator). Keep the tray in the incubator when not in use. Insert the slides into the incubation tray.
30m
Add ~5 drops RNAScope Protease Plus (Protease III-Cheadle) to cover each section and place tray into the incubator at40 °C for00:30:00 (standard) (00:15:00 -Otero-Garcia).
Note
Prepare RNAscope assay reagents during this step.
45m
Wash slides with 200 mL+ distilled water and slight agitation.
Wash slides with 200 mL + distilled water and slight agitation. (1/2)
Wash slides with 200 mL + distilled water and slight agitation. (2/2)
RNAscope Multiplex Fluorescent v2 Assay (Day 2): Preparation
RNAscope Multiplex Fluorescent v2 Assay (Day 2): Preparation
1h 15m
1h 15m
Wash Buffer: Warm 50x Buffer to 40 °C for 00:10:00 to00:20:00 . Add 980 mL distilled water to 20 mL of RNAscope Wash Buffer in a 1 L bottle. May need 1 L to 2 L per run. Mix well. Can be stored for up to one month.
30m
Probes: Prepare only those probes needed.
Note
If you are only using C2 and C3, dilute in probe diluent instead of C1.
Warm probes for 00:10:00 at 40 °C , then let cool toRoom temperature . Add 1 volume C2 and volume C3 probes to 50 volumes C1 probe in a tube (e.g. 200 µL C1 + 4 µL C2). Invert to mix. Store at 4 °C for up to 6 months.
10m
Reagents: Warm AMP1-3, HRP-C1-3 and HRP blocks at Room temperature .
(Optional) Saline Sodium Citrate: 175.3 g NaCl + 88.2 g sodium citrate in 800 mL istilled water. Adjust to7.0 with 1 Molarity (M) HCl. Add water to a final volume of 1 L . Sterilize by autoclaving and store at Room temperature for up to 2 months.
RNAscope Multiplex Fluorescent v2 Assay (Day 2): Hybridize Probes
RNAscope Multiplex Fluorescent v2 Assay (Day 2): Hybridize Probes
1h 15m
1h 15m
Remove liquid from slides. Add 4-6 drops (6 drops=180 µL ) of the probe mix to slides. Incubate in incubator for 02:00:00 at 40 °C .
2h
Wash slides with Wash Buffer.
Wash slides with Wash Buffer 00:02:00 at Room temperature . (1/2)
2m
Wash slides with Wash Buffer 00:02:00 at Room temperature . (2/2)
2m
OPTIONAL PAUSE POINT: Store slides in 5x SSC Overnight at Room temperature .
2m
RNAscope Multiplex Fluorescent v2 Assay (Day 2): Hybridize AMPs
RNAscope Multiplex Fluorescent v2 Assay (Day 2): Hybridize AMPs
1h 15m
1h 15m
Remove liquid from slides. Add 4-6 drops RNAScope Multiplex FL v2 Amp 1 to each slide. Incubate in incubator for 00:30:00 at40 °C .
30m
Wash slides with Wash Buffer.
Wash slides with Wash Buffer 00:02:00 at Room temperature . (1/2)
2m
Wash slides with Wash Buffer 00:02:00 at Room temperature . (2/2)
2m
Repeat steps 22 and 23 for Amp 2 and Amp 3. Amp 3 only requires 00:15:00 at 40 °C .
15m
During this incubation, dilute necessary Opal Dye fluorophores in TSA Buffer (1:1500 standard).
RNAscope Multiplex Fluorescent v2 Assay (Day 2): Develop HRP Signals
RNAscope Multiplex Fluorescent v2 Assay (Day 2): Develop HRP Signals
1h 15m
1h 15m
Remove liquid from slides. Add 4-6 drops RNAScope Multiplex FL v2 HRP-C1 to each slide. Incubate in incubator for 00:15:00 at 40 °C .
15m
Wash slides with Wash Buffer.
Wash slides with Wash Buffer 00:02:00 at Room temperature . (1/2)
2m
Wash slides with Wash Buffer 00:02:00 at Room temperature . (2/2)
2m
Remove liquid from slides. Add 200 µL Opal 520 to each slide. Incubate in HybEZ Oven for00:30:00 at 40 °C .
30m
Wash slides with Wash Buffer.
Wash slides with Wash Buffer 00:02:00 at Room temperature . (1/2)
2m
Wash slides with Wash Buffer 00:02:00 at Room temperature . (2/2)
2m
Remove liquid from slides. Add 4-6 drops RNAScope Multiplex FL v2 HRP Blocker to each slide. Incubate in incubator for 00:15:00 at 40 °C .
15m
Wash slides with Wash Buffer.
Wash slides with Wash Buffer 00:02:00 at Room temperature . (1/2)
2m
Wash slides with Wash Buffer 00:02:00 at Room temperature . (2/2)
2m
STOP HERE IF USING JUST C1 PROBE. Continue to Immunofluorescence.
Repeat steps 26-32 with HRP-C2 and Opal 570, and again with HRP-C3 and Opal 690.
Note
*Note that after additional of fluorophores, slides should be kept out of the light as much as possible.
4% PFA fix for 00:15:00 at 4 °C .
15m
Then, wash with PBS-Otero-Garcia for 00:04:00 . (1/2)
4m
Wash with PBS-Otero-Garcia for 00:04:00 . (2/2)
4m
Day 2: Immunofluorescence
Day 2: Immunofluorescence
1h 15m
1h 15m
Wash in0.1 Molarity (M) Tris buffer, 7.6 00:05:00 . Discard all Tris washes.
5m
Block in 0.1 Molarity (M) Tris/2% FBS (Tris/FBS) 00:30:00 +. Keep blocking solution for up to 2 weeks @ 4 °C .
30m
Dilute primary antibodies in Tris/FBS), and prepare humidified chamber(s) by soaking towel in the middle of the slide chamber(s).
Wipe excess fluid off back of slides and from around tissue and apply200 µL of primary antibody to slides.
Incubate at4 °C in humidified chamber00:45:00 to 02:00:00 at Room temperature or Overnight at 4 °C .
4h 45m
Day 3
Day 3
1h 15m
1h 15m
Rinse off antibody from tissue using Tris.
Note
Carefully direct spray from wash bottle around tissue, NOT directly on it.
Wash in Tris 00:05:00 .
5m
Block in Tris/FBS 00:05:00 .
5m
Dilute fluorophore-conjugated secondary antibody 1:500 in Tris/FBS and apply 200 µL to wiped slides. Incubate atRoom temperature for 02:00:00 or Overnight at 4 °C .
4h
Rinse off slides with Tris.
Wash in running tap H2O for 00:05:00 .
5m
Wash in Tris for 00:05:00 in green boats.
5m
Coverslip using non-photobleaching reagent (Prolong Gold with DAPI or FluorMount with DAPI). Allow to dry completely before imaging on scanner.