Oct 13, 2024

Public workspaceRNA Fluorescent in situ hybridization (FISH)

DOI
dx.doi.org/10.17504/protocols.io.14egn63dpl5d/v1
  • 1Washington University
Cecilia Escudero
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DOI: dx.doi.org/10.17504/protocols.io.14egn63dpl5d/v1
Protocol Citation: Carolina Lopez 2024. RNA Fluorescent in situ hybridization (FISH). protocols.io https://dx.doi.org/10.17504/protocols.io.14egn63dpl5d/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: May 28, 2024
Last Modified: October 13, 2024
Protocol Integer ID: 100751
Abstract
Protocol for RNA Fluorescent in situ hybridization (FISH)
Materials


Materials:
1. Nuclease Free water (NF H2O)
2. Formamide
3. 20X SSC and 2X SSC
5. PBS
9. 70% EtOH
10. Probe: 1 μL/reaction
* Probe prep: Stock (Concentration12.5 micromolar (µM) diluted in TE buffer)
SeV probes: 1:10 working solution (1.25uM) diluted in H2O, Amount5 µL stock + Amount45 µL UltraPure H2O, in vivo only
SeV probes: 1:100 working solution (125nM) diluted in H2O, Amount5 µL 1:10 + Amount45 µL UltraPure H2O, in vitro only
RSV probes: 1:50 working solution (250nM) diluted in H2O, Amount10 µL 1:10 + Amount45 µL UltraPure H2O, in vitro only

Solutions:
1. Fixation solution (4% FA):
(prepare fresh) Amount5 mL 37% formaldehyde (100% formalin),
Amount45 mL PBS 1X
2. Wash buffer:
(prepare fresh) Amount5 mL 20x SSC
Amount5 mL formamide
Amount40 mL NF H2O

3. 70% EtOH: Amount70 mL 100% EtOH,
Amount30 mL NF H2O

4. Hybridization buffer: 1 g dextran sulfate in Amount7 mL NF H2O (mix by rotation, take half an hour to dissolve).
Amount1 mL formamide
Amount1 mL 20x SSC
Volume up to Amount10 mL
* Store at Temperature-20 °C in Amount500 µL aliquots; good for years.

5. Mounting media: ProLong Diamond Anti-fade Mountant (Cat #P36961)


For GLOX anti-fade:

Anti-fade buffer: Amount850 µL Nuclease-Free H2O
(per slide) Amount100 µL 20x SSC
Amount40 µL 10% w/v glucose
Amount10 µL Tris-HCl (Ph8 )

Anti-fade solution: Amount100 µL Anti-fade buffer
Amount1 µL glucose oxidase
Amount1 µL catalase
Equipment:
Equipment:
Hybridization chamber:
How to mount the hybridization chamber.

Protocol:
Protocol:
Before starting, make sure to clean everything with RNaseZAP (Cat # R2020-250ML) and ethanol (i.e. gloves, pipettes, surfaces, etc) and always use filter tips.

Fixation and Permeabilization
1) Aspirate growth medium, and wash with Amount1 mL of 1X PBS.
2) Add Amount1 mL of fixation solution.
3) Incubate at room temperature for 10 minutes (you can move from tissue culture hood to your bench after this step).
4) Wash 2X with PBS.
5) Permeabilize cells in Amount1 mL of 70% ethanol for at least 1 hr at RT. Or O/N at Temperature4 °C .
(Cells can be stored at Temperature4 °C in 70% ethanol up to a week before hybridization).

Hybridization
— If frozen before using, thaw the reconstituted probe solution to room temperature. Mix well by vortexing, then centrifuge briefly.
— To prepare the hybridization solution, add Amount1 µL of probe working solution to Amount50 µL of hybridization buffer, and then vortex and centrifuge. *Final concentration of the probes should be tested and optimized.
1) Aspirate the 70% ethanol off the coverglass.
2) Add Amount1 mL of wash buffer, and incubate at RT for 2-5 minutes.
3) Prepare hybridization mix and assemble humidified hybridization chamber: see equipment
4) Add Amount50 µL of the hybridization buffer containing probe onto the parafilm.
5) Gently transfer the coverglass, cells side down, onto the hybridization solution. Cover the hybridization chamber (see equipment section) with the tissue culture lid. Incubate in the dark at Temperature37 °C for at least 4 hours, better overnight.

Wash
1) Transfer the cover glass, cells side up, to a fresh plate containing Amount1 mL of wash buffer, immediately.
Incubate in the dark at Temperature37 °C for 30 minutes.
2) Wash with Amount1 mL wash buffer consisting of 5 ng/mL DAPI to counterstain the nuclei.
Incubate in the dark at Temperature37 °C for 30 minutes.
3) Wash with Amount1 mL of 2X SSC. Incubate at room temperature for 2-5 min.
  1. 4) Mounting slides: Put a drop of ProLong Diamond Antifade (Cat # P36961) on to slide, dry off slide using aspirator and flip cell side down on to mounting media, make sure there are no bubbles. Mounting media needs to “cure” to correct refractive index overnight at room temerature (in dark), then can be moved to slidebox/proceed to imaging.
  • Alternatively, GLOX anti-fade may be used if necessary/want to image right away. In this case, seal the coverglass perimeter with rubber cement, and allow to dry. Store slides on ice. If necessary, gently wipe away any dried salt off the coverglass with water. Proceed directly to imaging.
Lopez Lab Specifics
Lopez Lab Specifics
Probe preparation for hybridization:
When adding Amount1 µL of each probe in Amount50 µL of hybridization buffer, the final working probe solution should be 125 nM for SeV probes and 250 nM for RSV probes. Adjust depending on the probe set, virus and optimized dilution tested.
* It is always recommended to have immunofluorescence slides staining for viral protein as controls for efficient infection.

Protocol references
Updated by: Lavinia Gonzalez 03/31/23

Protocol adapted from BioreserachTech. For the original protocol: https://www.biosearchtech.com/assets/bti_stellaris_protocol_adherent_cell.pdf