May 20, 2025
FISH CODETECTION
- Sylvie Dumas1,
- Åsa allén-Mackenzie1
- 1Lund University
Protocol Citation: Sylvie Dumas, Åsa allén-Mackenzie 2025. FISH CODETECTION. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6y631vqe/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 19, 2025
Last Modified: May 20, 2025
Protocol Integer ID: 218572
Keywords: ASAPCRN, riboprobes fluorescent in situ, riboprobes fluorescent, fluorescein, fish codetection dig, brain section
Funders Acknowledgements:
ASAP
Grant ID: ASAP-025188
Abstract
DIG and Fluorescein labeled
riboprobes
Fluorescent in situ
hybridization
Frozen brain sections
Image Attribution
Troubleshooting
PCR production by plasmid of interest
PCR reagents/plasmid
- In ice, place in a PCR tube :
H2O: 21µl
Mix primers S et AS (10 µM each) : 3 µl
DNA plasmid (10ng/µl): 6 µl
REDEXTRACT PCR Mix : 30µl
Dispatch 2 times 19,8uL of PCR mix in two other PCR tubes
(Note that the optimal vol for this PCR is 20 µl).
PCR Cycles
94ºC 5 min
94ºC 45sec
x 35 55ºC 45 sec
72ºC 1 min
72ºC 5 min
4ºC …
PCR gel control:
Make a 1% agarose gel
-1 µl 1kb+DNA ladder+ lµl of10X Blue juice Gel loading buffer + 8 µl H20
-5 µl of PCR product/ mRNA
PCR purification, (QIAquick PCR Purification
kit)
o Pool the 3 PCR tubes in an 1,5 ml tube
o Add 5 volume PB buffer to 1 volume of the PCR reaction (roughly 250 µl de PB) and mix
o Place a QIAquick column in a provided 2 ml collection tube
o To bind DNA, apply the sample to the QIAck column.
o Centrifuge
1 min at 13 000 rpm
o Discard flow-through
o Place the QIAquick column back in the same tube
o To wash, add 750 ul of PE Buffer to the QIAquick column
o Centrifuge 1 min à 13 000 rpm .
o Discard flow-through
o Place the QIAquick column back in the same tube
o Centrifuge 1 min à 13 000 rpm to remove residual wash buffer.
o Discard flow-through
o Place the QIAquick column in a clean 1,5 ml RNAse free tube
o To elute DNA, add 50 µl Buffer EB to the center of the QIAquick membrane and wait for 3 min.
o Centrifuge 1 min à 13 000 rpm.
Dosage : Nano drop
Riboprobe synthesis
Transcription reagents/riboprobe
In ice, place in a 1,5 ml tube :
5X transcription buffer 4µl
10X Dig-RNA labelling mix
or 10X Fluorescein-RNA labelling mix 2µl
100mM DTT 1µl
Rnasin ( 20U/µl) 0.5µl
T3/T7 RNA
polymerase (20U/µl) 2.0µl
PCR-product 150 ng
H2O up to 20 µl
Transcription reaction
Incubate 2h at 37°C
Riboprobe purification (illustra ProbeQuant G-50
Micro Columns)
Column preparation
- Re-suspend the resin in the column by vortexing
- Loosen the cap one-quarter turn and twist off
the bottom closure
- Place the G-50 column in the supplied
Collection tube
- Centrifuge 1 minute 735 × g
- Place the G-50 column in a clean 1,5 ml RNAse
free tube
Sample application
- Add to the riboprobe sample 30 µl Probe Buffer
type 1.
- apply the sample in the middle of the column.
-
Centrifuge 2 min à 750 g at 4°C
-
Place tube in ice
Dosage
(Nanodrop)
Storage
- Add 100 µl
Hyb Buffer (50% « Hybe–F » + 50% Formamide).
- Aliquot in 100 µl tube (50 µl/tube)
- Keep at -80°C.
Tissue Preparation
Adult mice are sacrificed by cervical dislocation, and brains are rapidly dissected out and snap frozen in cold (-30°C to -35°C) 2-methylbutane (≥99%, Honeywell, M32631) on dry ice. Brains are sealed with parafilm and aluminum foil and stored at -80°C until use. Embryonic brains, which are less rich in myelin and fat,
require more gentle freezing in 2-methylbutane (-20°C to -25°C) immediately after extraction from the euthanized and sacrificed mother.
Hybridization
-1- Thaw slides (around 5 min)
-2- Fix slides in 4% PFA in PBS, 10 min at RT.
-3- Wash 3x10 min inPBS
-4- Acetylation treatment:
- Equilibrate slides in TEA 5 min
- Prepare extemporaneously the acetylated buffer in the following ratio:
acetic anhydride 25 µl in 10 ml TEA
- Acetylation treatment, 10 min at RT
- Wash 3x5 min in PBS
-5- Hybridization:
- Make the Hyb Buffer by mixing Hyb Buffer
without Formamide + Formamide (50/50). Keep it on ice.
- For each riboprobe. Add to 20 µl Hyb Buffer the 2 riboprobes. Amount in accordance with the number of slides that will be hybridized:
o Dig-probe (50-75ng/100µl)
o Fluoresceinprobe (75-100 ng/100 µl)
- Denature 10 min at 85 °C
- Put in ice immediately 1 min
- Briefly centrifuge tubes at 2000g
- Put in ice
- Add XXX µl of cold Hyb Buffer adapted to the number of slides that will be hybridized.
Keep tubes in ice
- Vortex
- Take slide one by one from the PBS, remove the PBS by shaking the slide, dry the bottom of the slide with a klennex. Place slide in 3 slides hybridization box.
- Put 150 µl of (Hyb Buffer+ denaturated riboprobe) /slide
- Cover with a RNAse free coverslip (Hybrislip)
- Place the 3 slides hybridization box in a humidified chamber (5xSSC)
Note: no need to put Formamide here!
- Hybridize at 65°C between 16h and 18h.
- Prewarm X5 SSC and 0,2X SSC wash solutions at 65°C
Day 2: washes and immunological staining
- Place slide one by one in prewarmed X5 SSC and remove the coverslip
- Transfer slides in prewarmed X5 SSC and wash 5 min at 65°C
- Wash 3x20 min in prewarmed 0.2X SSC at 65°C
- Wash 10 min in 0.2X SSC at RT
- Wash 3 x 10min in MABT (RT)
From now, all steps at RT
Fluorescein-riboprobe
revelation.
- Place slides in humidified chamber (H20)
- Add 500 µl of BR/slide for 20 min
- Replace solution by 1/5000 anti-Fluorescein-POD in BR (500µl/lame) for 1h30
- Wash 3x 10 min in MABT
- Wash 3 x 10 min PBST
During the last wash, prepare a H2O2 0,001% solution from a 30% H2O2 by cascade dilution:
- 10µL H2O2 30% + 90µL PBST H2O2 3%
- 10µL H2O2 3% + 590µL PBST H2O2 0.05%
- 10µL H2O2 0.05% + 490µL PBST H2O2 0.001%
- From now everything must be done away from light.
- Place slides in humidified chamber (H20)
- Place the solution 1/250 Fluorescein-tyramide in H2O2
0.001% (500 µl/slide)
- When all slides are covered, gently move the
slide for a uniform distribution of the solution at the slide surface
- Incubation 1h at RT
- Wash 3 x 10 min PBST
POD- inhibition:
- Wash 20 min in Glycine buffer 0,1M pH= 2.1
- Quick wash in PBS
- Wash 20 min in 3% H2O2 in PBS
- Wash 3 x10 min in PBST
- Keep the slides in the last wash at 4°C
overnight
Day3: washes and immunological staining
Dig-riboprobe revelation:
- Wash 1 x10 min in PBST
- Wash 3 x 10 min MABT
- Place slides in humidified chamber (H20)
- Add 450 µl of BR/slide for 20 min
- Replace solution by 1/1500 anti-DIG-POD in BR (450µl/lame)
for 1h
- Wash 3x 10 min in MABT
- Wash 3 x 10 min PBST
During the last wash, prepare a H2O2 0,001% solution
- Prepare 0.001% H2O2 from 30% H2O2 by cascade
dilution:
- 10µL H2O2 30% + 90µL PBST H2O2 3%
- 10µL H2O2 3% + 590µL PBST H2O2 0.05%
- 10µL H2O2 0.05% + 490µL PBST H2O2 0.001%
- Place slides in humidified chamber (H20)
- Place the solution 1/250 Tamra-tyramide in H2O2 0.001% (500 µl/slide)
- When all slides are covered, gently move the
slide for a uniform distribution of the solution at the slide surface
-
Incubation 1h at RT
- Wash 5 x 10 min PBST
- Place slides in humidified chamber (H20)
- Add 450 µl/slide of DAPI for 20 min
- Wash 3x 5min PBST
- Mount in fluoromount.
- Quick microscope slide analysis
Give slide to be scanned