FLUORESCENT IN SITU HYBRIDIZATION (FISH) and post-FISH IMMUNOFLUORESCENCE on fresh frozen sections - ORAMACELL PROTOCOL

Sylvie Dumas; Véronique Bernard; Alice Abbondanza; Åsa Wallén-Mackenzie

Apr 23, 2026

Version 2

FLUORESCENT IN SITU HYBRIDIZATION (FISH) and post-FISH IMMUNOFLUORESCENCE on fresh frozen sections - ORAMACELL PROTOCOL V.2

DOI

https://dx.doi.org/10.17504/protocols.io.bp2l6y631vqe/v2

Sylvie Dumas¹,
Véronique Bernard²,
Alice Abbondanza^3,2,
Åsa Wallén-Mackenzie⁴

¹Oramacell, Paris, France;
²Sorbonne Université, CNRS, Inserm, Institut de Biologie Paris Seine (IBPS), Centre de Neuroscience Sorbonne Université (NeuroSU), 75005, Paris, France.;
³Laboratory of Cholinergic Signaling, Institute of Physiology of the Czech Academy of Sciences, Prague, 14200, Czech Republic;
⁴Lund University, Dept Experimental Medical Science, Molecular Neuroanatomy Lab, Biomedical Centre, 221 84 Lund, Sweden

Sylvie Dumas: [email protected];
Véronique Bernard: [email protected];
Alice Abbondanza: [email protected];
Åsa Wallén-Mackenzie: [email protected]

Lund University

Åsa Wallén-Mackenzie

DOI: https://dx.doi.org/10.17504/protocols.io.bp2l6y631vqe/v2

Protocol Citation: Sylvie Dumas, Véronique Bernard, Alice Abbondanza, Åsa Wallén-Mackenzie 2026. FLUORESCENT IN SITU HYBRIDIZATION (FISH) and post-FISH IMMUNOFLUORESCENCE on fresh frozen sections - ORAMACELL PROTOCOL. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6y631vqe/v2Version created by Åsa Wallén-Mackenzie

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 really well.

Created: April 21, 2026

Last Modified: April 23, 2026

Protocol Integer ID: 315423

Keywords: ASAPCRN, riboprobes fluorescent in situ, riboprobes fluorescent, fluorescein, fish codetection dig, brain section, Fluorescent in situ hybridization, FISH, histology, brain sections, mRNA, gene expression, spatial mrna analysis with protein localization, several mrnas in brain tissue, spatial mrna analysis, protein localization, oramacell protocol fluoresent in situ hybridization, detection of mrna, fluorescent in situ hybridization, mrna, several mrna, brain tissue, situ hybridization, fluorescent in situ, oramacell protocol fluoresent, brain section, high reliability in brain section

Funders Acknowledgements:

ASAP

Grant ID: ASAP-025188

Abstract

Fluoresent in situ hybridization (FISH) offers the advantage of localizing one or several mRNAs in brain tissue with high spatial resolution and extent of co-localization. The protocol presented here has been implemented in several studies and has allowed the detection of mRNAs with high reliability in brain sections and also other tissue material across several species. The protocol includes post-FISH immunofluorescent analysis, an optional step for combination of spatial mRNA analysis with protein localization. Note that the tissue should preferably be fresh frozen and cry-sectioned.

Keywords: 
 DIG and Fluorescein labeled
riboprobes
Fluorescent in situ
hybridization 
Fluorescent in situ
hybridization followed by fluorescent immunohistochemistry
Frozen brain sections

Image Attribution

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

Immunofluorescence protocol (post-FISH)

- Incubate primary antibody in FISH blocking buffer overnight at RT. 
- After washes in maleic buffer, 0.1% MABT and PBS, incubate slides with biotinylated goat anti-mouse igG   antibody (Vector Laboratories #BP-9200) diluted 1:400 in PBS, 1 h at RT. 
- Then wash in PBS and incubate in streptavidin-HRP (Vector Laboratories #SA-5004) in PBS for 1 hr. 
- Revelation of signal is done using Cy2-tyramide (from the FISH protocol) at a dilution of 1:250. 