Feb 25, 2026
“Green” WHOLE-MOUNT IN SITU HYBRIDIZATION/ Immunostaining/TUNEL on zebrafish embryos
- Gaia Gestri1,
- Kate Turner1
- 1UCL
- FishFloorUCL
Protocol Citation: Gaia Gestri, Kate Turner 2026. “Green” WHOLE-MOUNT IN SITU HYBRIDIZATION/ Immunostaining/TUNEL on zebrafish embryos. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgboxp3lpk/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: January 13, 2026
Last Modified: February 25, 2026
Protocol Integer ID: 238533
Keywords: green, sustainability, plastic, in-situ, immuno, TUNEL, zebrafish, various lab plastic, lab plastic, tunel on zebrafish embryo, zebrafish embryo, plastic waste, reusable pipette, use of plastic tip, plastic tip, less plastic, laboratory visit
Funders Acknowledgements:
MRC
Grant ID: MR/T020164/1
Abstract
We want to encourage other scientists to reduce and reuse lab plastic as much as possible in their experiments. Here we present our standard in-situ/immuno/TUNEL protocols with specific advice on how we minimise our use of plastic tips, pipettes and tubes-with no negative repercussions for our experiments.
We include lots of images for how we organise and store our various lab plastics so we can use them again and again. You will be amazed at how few fresh tips you need for your experiments. If everyone followed this guidance the reduction in plastic waste generated in the life sciences would be huge!
Please note the reason we made this protocol is to encourage you to use less plastic so it is by no means the shortest version of these protocols. Once you are set up with your reusable pipettes and tips you can switch to using the normal protocols.
For more tips on increasing the sustainability of your laboratory visit our website https://zebrafishucl.org/#zebrafish-green-lab
Guidelines
General tips to reduce plastic:
- Consider that smaller tips use less plastic that bigger one e.g. 200μl tips are way smaller than 1000ml so, if you have to collect 250μl you could pipette 125μlx2.
- Make aliquots of pK in small PCR tubes
- Fix embryos in small tubes-Eppendorfs or small glass vials and not falcons or big plastic containers.
- Use glass when possible to store your solutions.
- Have a dedicated box with tips/or container with Pasteur pipettes that you use for these protocols
- Store tips and pipettes inside or with the solutions they are for (see our photos for the different ways of doing this).
- Every tip can be re-used multiple times in these protocols; we tend not to reuse the tips used for the probes just because we tend to do in-situ with lots of different probes every time. If you do in situ with the same probes over and over again (e.g-looking at the effect of different concentration of a drug on a particular gene – you could keep your probe tip in a special box together with the probe).
Materials
Solutions:
Hyb(+) -- use for prehyb and hyb
Formamide (Sigma f7508) 25 ml
20X SSC 12.5ml
50mg/ml Torula RNA (Sigma R6875) 500μl
100 mg /ml Heparin (Sigma H8537) 25μl
20% Tween-20 250μl
1M Citric acid 460μl
H2O 11.27ml
Store at -20˚C
Hyb(-)
Formamide(Sigma f7508) 25 ml
20X SSC 12.5ml
H2O 12.5 ml
SSC = sodium citrate:
20X SSC (1litre)
175.3g NaCl (3M, MW 58.44)
88.2g TriSodium citrate (0.3M, MW 294.10)
Top up to 1litre with H2O
MABlock = Maleic Acid
Buffer + 2% Roche Blocking reagent:
Maleic Acid Buffer pH 7.5 (800ml)
7g NaCl(MW-58.44)
9.3g Maleic Acid (MW-116.1)
Top up to 800ml with H20, adjust to pH7.5; [Final] = 150 mM NaCl, 100mmMalic Acid
AP Buffer (50ml) =buffer for alkaline phosphatase activity:
Notes: Make fresh each time!
You can easily scale back the recipe to 10 or 20 ml so no need to waste it.
The pH of this solution is very important.
We make a TRIS PH 9.5 fresh every 3 months mixing together TRIS HCL with TRIS BASE (to make 10ml of 1M TRIS PH 9.5= add 0.56ml of 1M TRIS HCL and 9.44 ml of 1M TRIS base see picture
1M Tris-HCL, pH=9.5 5 ml
1M MgCl2 2.5 ml
4M NaCl 1.25 ml
20% TX-100 (or Tween-20) 0.25 ml
H2O 41 ml
Troubleshooting
Before start
The first steps of the in-situ, immuno and TUNEL protocols are the same. So we will describe them together.
For immunostaining follow step 1-14 and then go to the dedicated immunostaining section of the protocol (STEP 28).
FIX EMBRYOS
STEP 2-STEP 14 are the same for all 3 protocols In-situ/TUNEL/Immunostaining
Fix embryos in 4% PFA for 3h at RT or overnight at 4˚C. This is generally valid for in-situ/TUNEL and most Abs.
* Some antibodies are more problematic and do work better with shorter fixation.
Make either a 20X or a 4X PFA stock in a glass bottle - we store the PFA stock at -20 and the working solution at 4 degrees for up to 1 week (see Fig 1).
Fig 1
Put zebrafish embryos in a 1.5ml Eppendorf and remove the extra fish water. Have a passette/tip dedicated to fish water (FW) that you can reuse again and again (see Fig 2).
Add 4%PFA with either a dedicated 1000ml PFA tip or a dedicated PFA pipette to be used over and over for all your in-situ/immuno/TUNEL experiments
Fig 2: Reusable labelled pipettes.
DEHYDRATE
Remove the PFA from the tube with your PFA tip/pipette.
Add methanol(MeOH) with either a 1000ml MeOH tip/or a MeOH pipette to be used over and over (see Fig 2).
Wash 2x with 100% MeOH (Store embryos at -20˚C at least overnight or can be stored up to few years)
REHYDRATE
Rehydrate your embryos into PBST series (PBS + 0.5-0.8% tween20 or triton100): 50%PBST/MeOH
5’, PBST 2x5’.
At the start of this step the embryos are in 100% MeOH. So remove 50% of MeOH with dedicated MeOH tip and add roughly same amount of PBST with dedicated PBST tip. Mix well by inverting the eppendorf tube.
*Make PBST in a glass bottle (see Fig 3)
Fig 3: Plastic pipette stored inside the glass bottle of solution.
or keep a reusable pipette attached see Fig 4
Fig 4: Side holster for plastic pipette.
We use Tween as a detergent for in-situ and TUNEL and Triton for immunostaining. These detergent are very viscous to work with so we tend to make a 10% stock solution. Tips can be stored in a box see Fig 5
Fig 5: Pipette tip box with tips and lid labelled for reuse.
Remove the 50%PBST/MeOH with a PBST/MeOH tip (to be reused FIG 5) and add PBST with your PBST tip or pipette (Fig 3 or 4) that you will use over and over for all the following washes (and many future experiments.)
PERMEABILISE
Digest with ProteinaseK: in 1x PBST (PBST)
* make aliquots in small PCR tubes instead of bigger Eppendorf tubes
- reuse your PK tip (see Fig 6).
*Store your dedicated PK tip either inside the box of aliquots or on your bench)
Fig 6: Storing PK tip for reuse.
Digestion Times for PK
Developmental Stage PK Treatment
Up to tailbud no PK
2 - 10ss quick rinse in 1X PK
10 - 15ss 1 min 1X PK
16 - 26ss 2 min 1X PK
24 hpf 15 min 1X PK
30 hpf 20 min 1X PK
36 - 48 hpf 30 - 40 min 1X PK
2.5 dpf 30 - 40 min 1.5X PK
3 dpf 30 - 40 min 2X PK
4 dpf 30 - 40 min 3X PK
5 dpf 30 - 40 min 4X PK
Note: If the embryos are dissected (for immunos), reduce the incubation time or avoid this step.
TUNEL: For better TUNEL staining always opt for a longer incubation time or higher concentration of PK. We haven’t tested all stages but have had good results for 30’ treatment in 24hpf embryos; 1h in 2X for 48hpf ; 1h in 3X PK for 3dpf. As a rule of thumb always opt for at least 1/3 longer incubation time and for 1X higher concentration of PK after 48hpf.
Remove the PK/PBS solution (with your PBST tip)
Refix with 4% PFA for 20’ at RT (with the PFA tip)
For this post fixing you don’t need a very fresh PFA-the protocol works well even if your PFA is 10 days old or similar.
Wash with PBST 4x5’ (with PBST tip)
*** PROTOCOLS DIVERGE ****
The in-situ hybridisation, TUNEL and immuno protocols start to vary so for TUNEL staining post permeabalisation please jump to STEP (22) and for immuno STEP (28)
In-situ Prehybridisation and Probe Incubation
Replace PBST with Hyb+ (enough to cover the embryos ~300 ul ) with your Hyb+ pipette tip
that you can leave inside the bottle at -20°C (see Fig 7 LEFT)
Incubate embryos at 68°C (heat block on its side or water bath) for at least 2h. Keep
the tip for the following step.
Remove Hyb+ (with Hyb+ pipette tip) and add add new Hyb+ to embryos (set the tip aside for the following day of washing), then dilute probes directly into the Eppendorf containing fresh
Hyb+ and embryos (mix well) and incubate overnight at 65 ˚C to 70˚C.
Fig 7: LEFT-Store HB+ pipette tip inside your glass bottle at -20°C.
RIGHT: Keep your 200 μl tip in the eppendorf you will store your working dilution to recover your probe the next day.
Notes: Heat blocks can reach the desired temperature quite quickly (it varies from heat block to heat block) so switch them on only 5/10’ before using them.
The dilution of the probe is always tip consuming: Dilutions of the probes can vary from time to time;
calibrate from single in situs; a good starting point is 1:200 -1:500. For a probe that you have not tested before you will need a new 10/20 μl tip per each probe to dilute in heat block (to be discarded) and a new 200μl tip to remove the probe the following day.
For a probe that you have a 1x working solution you will need a new 200 μl tip (NOTE: don’t throw it
away-keep it inside your 1x empty Eppendorf in order to reuse the following day
when you will be recovering the probe (see Fig 7 RIGHT). If you have space and you are well organised and very committed you will find ways to keep specific tips for different probes-see general advice.
DAY 2 In-situ ONLY: POST-HYBRIDISATION
Recover the probe (probe can be reused several times). If you had used a 1x probe solution you can use the tip from the previous day (see Fig 7 RIGHT). If you have been using a new probe you need to use a new 200 μl tip for each probe.
Wash 4x 30min in Hyb- at 68˚C (reuse your Hyb+ tip from previous day)
Wash 15’ in 2X SSC at 68˚C (We have one SSC pipette to use for both 2X or 0.2 X SSC) (see Fig 8).
FIG 8: In-situ reusable pipette collection. Stored together to make life easy!
Wash 2x15’ in 0.2X SSC at 68˚C (reuse tip)
Go to STEP 24 to continue in-situ protocol
TUNEL (DAY 1:Continued from STEP 14)
Replace PBST with pre-chilled EtOH:Acetone 2:1 (keep the bottle with at -20˚C, with the
tip inside it (see Fig 9).
FIG 9: LEFT a 1ml tip fits perfectly inside a 50ml bottle.
RIGHT: Tips for reaction buffer and Tdt can be labelled and reused. We store in the freezer -20˚C alongside the reagents themselves.
Put the embryos at -20˚C for 10’
Remove EtOH:Acetone with your EtOH:Acetone tip rinse with PBST 3x5’ at room temperature (use your PBST tip or pipette).
TdT REACTION
Replace PBST with equilibration buffer (from ApopTag) 1h at room temperature (use a 1ml equilibration buffer tip from the tip box-see Fig 5).
Meanwhile prepare the enzyme reaction: Reaction buffer 24 μl :Tdt 12 μl : 1ul of 10% triton.
Given that the ratio of reaction buffer to enzyme is 2:1 try to use the minimum amount of enzymatic reaction as possible. 37 μl in total is enough to cover 10/15 embryos up to 48 hpf. Take off as much equilibration buffer as possible (with the equilibration buffer tip-put the tip back in the box FIG 9 RIGHT) but still leave enough to cover the embryos. And add enzymatic reaction.
***Tips for reaction buffer and Tdt can be labelled and reused, we store them in the freezer box with the rest of the TUNEL kit (see Fig 9 RIGHT)***
Incubate at 37 ˚C O/N (tube vertical in water bath).
DAY 2: TUNEL ONLY
Wash in PBST at 37 ˚C for 1h changing the solution several times (with the usual PBST tip)
DAY 2 ***In-situ and TUNEL***
Wash 4x15’ in PBST at RT (reuse PBST tip)
Block in MABlock for at least 2h at RT (new tip here)
Ab Staining for In-situ and TUNEL
MAB blocking + Ab (anti FLUO 1:4000 or anti DIG 1:6000; Roche) overnight at 4°C.
*Ab tips can be stored and reused (see Fig 10)
FIG 10: Store communal Ab tips with the antibodies at 4°C.
DAY 3- In-situ and TUNEL: Washing and Colour Development
Wash with PBST; 2 quick rinses then 4x30’ (reuse tip)
Wash with freshly prepared AP buffer (See Materials section attached to this protocol)– 1x10’ (keep reuseable tip in your tip box see FIG5)
Incubate embryos in AP Buffer + NBT/BCIP in the dark at RT (lμl NBT + 3.5μl BCIP per 1μl of AP Buffer)
*NBT/BCIP tips can be stored in the box (see Fig 11).
FIG 11: NBT/BCIP tips can be stored in the box with the reagent and used communally.
Use 400-500 μl of solution per tube. We used to transfer embryos from Eppendorf tubes into plastic wells for the colorimetric reaction. To reduce plastic usage we now develop in the same eppendorf tubes and we check reaction using a glass well.
Development time varies from 30’ to a week *Watch out for background when leaving embryos for long times, you can slow down the reaction putting the embryos at 4°C or diluting the NBT/BCIP in AP buffer
or speed up the reaction by moving the embryos at warmer temperatures e.g. 30°C incubator
When reaction is finished remove the staining solution with the NBC/BCIP tip and rinse in AP
buffer (same AP buffer tip)
fix overnight in 4% PFA at 4˚C (use same PFA tip)
*for post fix of the colour, if you have any PFA that is older than 1 week is OK to use it for this step as freshness is not critical for this step.
TUNEL staining develops very rapidly with the standard 1X NBT/BCIP mix; therefore a 0.5X
dilution of NBC/BICIP to slow colour development is recommended. Alternatively, a 1:10.000 dilution of the anti:DIG Ab instead of the 1:6000 suggested by standard protocols also helps control the speed of the reaction)
IMMUNOSTAINING (Continued from STEP 14)
DAY 1: Blocking and Primary Ab Incubation
Remove PBTr (reuse tip) and Block in IB for at least 1h at RT on a shaker.
(IB=5-10% Normal Serum
* the normal serum used for blocking needs to be from the same host species as the secondary Ab, concentration might vary. We generally used goat secondary Ab and block with 10% NGS ,1%DMSO;PBTr.
Incubate in IB plus primary ab overnight at 4 degrees. Some Ab need to be super concentrated e.g. dilution 1:25 in that case just use enough solution to cover the embryos.
*Ab tip can be store to be re-used as for FIG 10 & FIG 12.
FIG 12: Communal Ab tips stored with their antibody. We do this for Primary and Secondary Abs.
DAY 2:
Remove primary Ab with a new tip (200 μl one would be best).
Wash at least 4x30’ in PBST at RT on shaker (use your 1ml PBTr tip or pipette)
Incubate in either PBTr or IB + secondary Ab from 2h at room temperature/ON at 4 °C (secondary Ab
tips can be stored (see Fig 12).
DAY 3:
Remove secondary Ab with either a new tip or tip from STEP 32
Rinse 3x in PBTr and wash at least 4 times x30’ in PBTr (you can use your usual PBTr tip or pipette).
Wash in PBS and either mount them in agarose or *wash stepwise into glycerol.
*Add glycerol to PBS to step down to increasing amounts of glycerol-5’ per concentration
Option 1 : have a dedicated bottle with 25% glycerol/PBS; 50% glycerol/PBS; 75% glycerol/PBS -you can use the same glycerol/PBS tip stored in your pipette tip box (see Fig 5)
Option2 :
- Leave circa 400μl of PBS in the tube with embryos and add 125 μl of glycerol -mix gently leave for 5’.
- Then add 375 μl of glycerol-mix well and leave it for 5’.
- Finally add another 400 μl of glycerol to complete transferer into glycerol/PBS (for option 2 you can have a glycerol tip attached to the glycerol bottle (see Fig 4).
Keep embryos in the dark and image as soon as possible.
Protocol references
The TUNEL protocol follows ApopTag Kit (Chemicon International) Manufacturer's instructions
TUNEL and In-situ hybridisation protocols were adapted from:
Q Xu, N Holder, R Patient, SW Wilson
Spatially regulated expression of three receptor tyrosine kinase genes during gastrulation in the zebrafish
Development, 1994
The Immunohistochemistry protocol was adapted from:
Turner KJ, Bracewell TG, Hawkins TA. Anatomical dissection of zebrafish brain development.
Methods Mol Biol. (2014) doi: 10.1007/978-1-62703-655-9_14.