Oct 13, 2024
IFA and FISH combined protocol
- 1Washington University
Protocol Citation: Carolina Lopez 2024. IFA and FISH combined protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk8or6l5r/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 24, 2024
Last Modified: October 13, 2024
Protocol Integer ID: 100597
Abstract
This protocol describes the procedures for staining preps with both RNA FISH and immunofluorescence
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 (12.5 micromolar (µM) diluted in TE buffer)
SeV probes: 1:10 working solution (1.25uM) diluted in H2O, 5 µL stock + 45 µL UltraPure H2O, in vivo only
SeV probes: 1:100 working solution (125nM) diluted in H2O, 5 µL 1:10 + 45 µL UltraPure H2O, in vitro only
RSV probes: 1:50 working solution (250nM) diluted in H2O, 10 µL 1:10 + 45 µL UltraPure H2O, in vitro only
Solutions:
1. Fixation solution (4% FA):
(prepare fresh) 5 mL 37% formaldehyde (100% formalin),
45 mL PBS 1X
2. Wash buffer:
(prepare fresh) 5 mL 20x SSC
5 mL formamide
40 mL NF H2O
3. 70% EtOH: 70 mL 100% EtOH,
30 mL NF H2O
4. Hybridization buffer: 1 g dextran sulfate in 7 mL NF H2O (mix by rotation, take half an hour to dissolve).
1 mL formamide
1 mL 20x SSC
Volume up to 10 mL
* Store at -20 °C in 500 µL aliquots; good for years.
5. Mounting media: ProLong Diamond Anti-fade Mountant (Cat #P36961)
6. For GLOX anti-fade:
Anti-fade buffer: 850 µL Nuclease-Free H2O
(per slide) 100 µL 20x SSC
40 µL 10% w/v glucose
10 µL Tris-HCl (8 )
Anti-fade solution: 100 µL Anti-fade buffer
1 µL glucose oxidase
1 µL catalase
Combine FISH with IFA
Combine FISH with IFA
When IFA and FISH are to be combined, we prefer to perform IFA (under RNAase-free conditions) prior to FISH, because the formamide treatment during the FISH procedure is sometimes incompatible with preservation of the epitopes detected by some antibodies.
Equipment:
Equipment:
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.
A. IFA:
1. Briefly rinse cells cultured on coverslips in sterile PBS.
2. Fix in filter-sterilized 4% formaldehyde for 00:10:00 at RT. (can move from TC hood to bench after this step)
3. Wash 3X in 8PBS.
4. Permeabilize with ice-cold 70% Ethanol for 01:00:00 (can leave O/N at 4 °C for up to a week)
5. Wash 3X in PBS.
6. Incubate with primary antibody diluted in 1% BSA in sterile PBS (2 µL RNAase OUT per 500 µL ) for 00:45:00 at RT in a dark and humid chamber.
7. Wash 3X in PBS.
8. Incubate with secondary antibody (diluted in the same solution as in step 6) for 00:40:00 at room temperature in a dark and humid chamber.
9. Wash at least 3X in PBS.
10. Post-fix in freshly made 4% formaldehyde for 00:10:00 at RT.
11. Wash cells with 2X SSC and then with wash buffer, 00:05:00 each.
B. FISH
--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 1 µL of probe working solution to 50 µL of hybridization buffer, and then vortex and centrifuge. *similar to anitbody concentration, probe concentration should be optimized for each probe set.
1. Aspirate the 70% ethanol off the coverglass.
2. Add 1 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 50 µL of the hybridization buffer containing probe onto the parafilm.
5. Gently transfer the coverglass, cells side down, onto the hybridization solution. Cover the humidified chamber with the tissue culture lid. Incubate in the dark at 37 °C for at least 04:00:00 , better O/N.
6h 50m
Wash
Wash
1) Transfer the cover glass, cells side up, to a fresh plate containing 1 mL of wash buffer, immediately.
Incubate in the dark at 37 °C for 00:30:00 .
2) Wash with 1 mL wash buffer consisting of 5 ng/mL DAPI to counterstain the nuclei.
Incubate in the dark at 37 °C for 00:30:00 .
3) Wash with 1 mL of 2X SSC. Incubate at RT for 2-5 min.
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 O/N at RT (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.
1h
Lopez Lab Specific Section:
Lopez Lab Specific Section:
Probe preparation for hybridization:
When adding 1 µL of each probe in 50 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: