May 20, 2025
Solutions used for FISH Codetection
- Åsa allén-Mackenzie1,
- Sylvie Dumas1
- 1Lund University
Protocol Citation: Åsa allén-Mackenzie, Sylvie Dumas 2025. Solutions used for FISH Codetection. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpqko1lzp/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 20, 2025
Last Modified: May 20, 2025
Protocol Integer ID: 218585
Keywords: ASAPCRN, fish codetection protocol, awm lab, protocol
Funders Acknowledgements:
ASAP
Grant ID: ASAP-025188
Abstract
protocol to make the solutions used in the protocol published by AWM lab for FISH Codetection
Troubleshooting
WATER
-1- MilliQ water directly pick up at the
source…
-2- nuclease free water
(Invitrogen AM9932)
PFA pH 7,5
40g in 900 ml H20 + 1 drop 10N NaOH
Heat for dissolution (no more than 65°C)
Then add 100 ml of x10 PBS
Filter 0,2 µm
Cool on ice for an immediate use.
Could be kept at 4°C 15 days without problem or at -20°C
TEA pH 8 (keep at RT)
13,3 ml TEA (MERCK)
Qsp 1 litre H2O
Adjust to pH=8.0 (with conc HCl)
HybBuffer: Hybridization buffer
Hyb Buffer without formamide 2 ml
- 20X SSC 10ml
- E.coli tRNA 50mg/ml 200 µl
- RNAse free water 4 ml
- X50 Denhardt’s 4ml
- Salmon Sperm DNA 10 mg/ml 2 ml
Aliquote in 2ml tube. Keep aliquots at -20°C.
These could be thawed and refreezed without pb!
Desionized Formamide
Important to use desionized formamide of good quality.
For this, formamide must be desionized in the lab, aliquoted (in 600 µl tube (500 µl/tube) or 1,5 ml tube
(1 ml/tube) and aliquots kept at -80°C
and not at -20°C! If you thaw a tube you should not refreeze it.
To deionize 500 ml formamide
- Weigh 25g resin AG501-X8
- Add 500 ml formamide (pH 7).
- Stir for 2 h.
- Verify pH to 5.5 (if the pH is still 7, discard the solution completely).
- Filter the solution with 0,22 µm nylon filter (do not use nitrocellulose filter!)
- Label the tubes with the content.
Freeze aliqiots to not be used immediately.
5X MAB (stock solution: keep at RT)
Maleic Acid: 58,04 g
NaCl : 43,82 g
Dilute in 850 ml H20 and adjust the pH to 7,5 using 10N (30%) NaOH
(for this add 90ml of NaOH then drop by drop. Note that the solution may stay turbid until the pH reach around 6,8).
Then qsp 1 liter with H20
MABT
MAB + 0,1% Tween20Untitled section
PBST
PBS + 0,1% Tween 20
BR (Blocking Reagent Buffer): keep at -20°C
For 200 ml
5x MAB : 40ml
FBS heat inactivated:40 ml
Blocking Reagent (Roche) 2 g
Qsp 200 ml H20
Make the solution and aliquote it in 15 ml tube. Could be thawed and refreezed.
Fluorescein-Tyramide
- Solution 1 : Make a 10 mg/ml
stock of the NHS ester in DMF (100 mg fluorescein NHS ester in 10 ml DMF)
- Solution 2 : Make DMF-TEA solution (4 ml DMF (dimethylformamide) + 40 ul TEA (triethylamine))
- Solution 3 : Make Tyramide solution (35 mg tyramide in 3,5 ml DMF-TEA (ie in solution 2)
To link the Fluorescein onto tyramide, the reaction is :
-
Mix 10 ml fluorescein-NHS in DMF (ie solution 1) + 3,43 ml tyramide solution (ie solution 3)
- Incubate in dark at room temp for 2 hours
- Then add 12 ml ethanol,
- Make aliquots of 500µl in 600µl tube store in dark at 4 or -20°C
Glycine 1M Ph2,1
For 100 ml:
- 7,5 g Glycine
- Add 90 mlH2O
- Adjust the pH to 2,1
- Qsp 100 ml H2O
- Aliquot in 10 ml tube
Keep aliquots at -20°C.
Dilute 10x in PBS before use.
DAPI
Stock: 5 ng/ml in water (this is kept in minus 20)
Dilute 1:25.000 in PBST (2 µl per 50 ml), keep at 4°C