Apr 06, 2023
Immunohistochemistry (IHC) Whole-Mount Antibody Staining
- 1Univeristy College London
Protocol Citation: Talia Pittman 2023. Immunohistochemistry (IHC) Whole-Mount Antibody Staining . protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8jzn8g2w/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
Created: April 06, 2023
Last Modified: April 06, 2023
Protocol Integer ID: 80104
Abstract
Immunohistochemistry (IHC) Whole-Mount Antibody Staining protocol @FishFloorUCL
Introduction
Introduction
Protocol based on Jenny Regan's GFP protocol, modified by Tom Hawkins thomas.hawkins@ucl.ac.uk
Fix and dehydrate
Fix and dehydrate
Depending on the antigen, you should either fix in PFA or TCA. Always start with PFA (option 1). TCA (option2) can be better for older embryos >36hrs
Either:
(Option 1) Fix in PFA
- Fix - Fix in sweet Paraformaldehydefix (4% w/v PFA 4% w/v sucrose in PBS pH 7.3) for 3 hrs at room temperature or 4°C overnight
- Wash - After fixing, transfer to 1.5 mL tubes and rinse 2x, and wash 3 x 10 mins in PBTr (Phosphate buffered saline + 0.5-0.8% Triton-X100) on a shaker
- (Optional) Dissect brains at this point
- Dehydrate - Transfer to MeOH
1 X 5 mins in 50% MeOH / 50% PBTr
2 X rinse in 100% MeOH
Store at 20°C for at least 30mins or for up to 6 months (good point to leave overnight)
Or:
(Option 2) Fix in TCA
- Fix - 2% TCA in PBS for exactly 3 hrs at room temperature in 5ml bijous
- Wash - Transfer to 1.5ml tubes, rinse 2x and wash 3 x 5mins PBS (on side)
Store at 4ºC for a week but add 20mM Azide if storing for longer (to prevent mould). In azide, PBS embryos should keep for a month.
(Day 1) Rehydrate
(Day 1) Rehydrate
This step applies to PFA fixed, MeOH stored embryos only
- 1 x 5mins 50% MeOH / 50% PBTr
- 3 x 5mins PBTr
(Day 1) Permeabilise
(Day 1) Permeabilise
For PFA:
- Digest at room temperature, with the tube lying on its side (no agitation) (use a 22ºC incubator in high summer or deep winter)
Store PK at -20°c at 10mg/ml – This is 1000x stock
ProteinaseK (PK) digestion times vary with embryo age. Digest according to the table below
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, reduce the incubation time or avoid this step.
2. Rinse 3x PBTr
3. Post fix in 4% PFA for 20 min at room temperature on gentle shaker
4. Wash 3x 5min PBTr
For TCA:
- Rinse embryos 3 x 5 mins PBS to remove azide.
- Prechill trypsin* solution (0.25% in PBS) and 5mL per tube of PBTr on ice until cold.
- Incubate embryos in trypsin in ice for 5-10 mins (according to age 36hpf - 5dpf) may need longer for older embryos, depending on the trypsin batch, titrate upon first use.
- Rinse 2x in cold PBTr then 3x10mins in cold PBTr, then bring to RT
*Trypsin stock is 2.5^% (10X)
(Day 1) Block
(Day 1) Block
Incubate in IB for at least 1 hour at room temp on shaker. (IB: 10% normal goat serum (NGS), 1% DMSO, 0.5-0.8% Triton-X100 in PBS)
- For 1ml IB: 100 mL NGS, 10 mL DMSO, 0.89 mL PBTr
- For 5ml IB: 500 mL NGS, 50 mL DMSO, 4.45 mL PBTr.
(Day 1) Apply Primary Antibody
(Day 1) Apply Primary Antibody
Incubate in IB + primary antibody overnight at 4°C on shaker
- AntiGFP: Polyclonal rabbit α-GFP from AMS Biotechnology (cat #TP401) gives great results. Use 1/1000. AMS Biotechnology, 185A/B Milton Park, Abingdon, Oxon OX14 4SR, UK.
- Titrate other antibodies upon first use. Monoclonals generally at between 1:100 and 1:1000 Supernatants at 1:5 - 1:50
(Day 2) Wash
(Day 2) Wash
- Remove the primary antibody (primary antibody can be kept at 4°C for reuse within a week)
- Rinse 3x in PBT.
- Wash at least 4 x 30min in PBTr on shaker.
(Day 2) Block Endogenous Peroxidases
(Day 2) Block Endogenous Peroxidases
Unnecessary with fluorescent-conjugated antibodies OR < 24hr embryos
- Wash 5 mins in 50% MeOH/PBTr
- Wash 10 mins in 100% MeOH
- Incubate in MeOH peroxide for 10 mins at room temperature (1 mL MeOH/10ml 30% H2O2)
- Wash 5 mins 50%MeOH/PBTr
- Wash 5 mins 50% MeOH/PBTr
(Day 2) Apply Secondary Antibody
(Day 2) Apply Secondary Antibody
Incubate in IB + secondary antibody overnight at 4°C on shaker
If you are using a fluorescently tagged antibody, keep tubes in the dark
Secondary depends upon primary antibody and detection method
- For fluorescence: Molecular Probes (www.probes.com) Alexa Fluor 488 goat α-rabbit IgG is good (cat # A-11034). Use at 1:200.
- For non-amplified HRP detection: Sigma (www.sigmaaldrich.com) goat α-rabbit IgG (whole molecule) peroxidase conjugate is good (cat # A-6154). Use at 1:200.
- For amplified HRP detection: Vector labs (www.vectorlabs.com) biotinylated anti-mouse IgG (#BA-9200) or anti-rabbit IgG (#BA-1000) are both great.
(Day 3) Wash
(Day 3) Wash
- Rinse 3 x in PBSTr
- Wash at least 4 x 30 min on shaker
If you are using a fluorescently tagged antibody, keep tubes in the dark
Fluorescently stained embryos are now ready to image.
- Rinse out Triton with PBS.
- Either transfer to 75% glycerol (+AF1 CITIFLUOR) (through 25% and 50% glycerol/PBS) and mount. Or keep in PBS and mount in Agarose.
Keep at 4°C in the dark and image as soon as possible.
(Day 3) DAB Detection
(Day 3) DAB Detection
See below for amplification for non-amplified detection (HRP conjugated secondary antibodies)
- Make up 1 mL DAB solution** per tube of embryos.
- Transfer embryos to a 24-well plate.
- Remove PBT and add 500ml DAB solution. Incubate for 20 min.
- Add 1 mL of 0.3% H2O2 per 1 mL of the remaining DAB solution.
- After the 20 mins is up, swap solution for 500ml DAB+ H2O2
Monitor reaction closely (can take 1min or 2hrs+, usually around 20-40 mins).
- More H202 can be added if it is slow.
- Always change the whole solution for a DAB solution with more H2O2 i.e. do not just add more H2O2to the solution with the embryos in it as it will cause some embryos to blacken instantly. Try 1 µl 0.6% per mL then 0.9% etc... this can go up to 6%, but this will usually cause background very high up, and you'd be better off using more or a better primary antibody.
DAB stock: Sigma D5637; 25mg/500ml dH2O. Keep aliquots at -20°c and thaw immediately before use.
**DAB solution: 500ml stock in 30ml PBS, 250µl in 15ml PBS, 125µl in 7.5ml PBS, 16.6µl per 1ml PBS
(Day 3) 'ABC' Amplification
(Day 3) 'ABC' Amplification
For amplified detection when using biotinylated secondary antibodies.
- Prepare ABC reagent: to 5ml of PBTr add 2 drops of vectorlabs elite 'A' reagent***, vortex.
- Allow to settle. Open the tube and pop any bubbles at the top of the tube and then add 2 drops of vectorlabs elite 'B' reagent, vortex and leave to stand for 20 mins.
- Incubate embryos in ABC reagent for 3-4 hours at room temperature on shaker or overnight at 4ºC on shaker.
- Wash as above (3 rinses then 4 x 30mins) and follow DAB detection section above
***Vectorlabs (www.vectorlabs.com) can provide basic ABC kit ('elite')(containing only A and B reagents Cat# PK-6100) or kits containing biotinylated antibodies and A & B reagents and blocking serum can also be bought (Cat #s anti-rabbit IgG: PK-6101anti-mouse IgG: PK-6102). The non-'elite' kit is probably fine too (use only one drop each of A and B, the elite probably makes not much of a difference anyway).
(Day 3) Wash
(Day 3) Wash
- Wash 3 x 10min in PBS.
- If embryos were not raised in PTU then here they can be bleached. For bleaching mix fresh bleach using 1 mL 10% w/v KOH 0.4ml 30% H2O2 topped up to 12.5 mL. Place embryos in solution and monitor. Bleaching usually takes about 45 mins. After which, rinse with PBS several times. If bleaching not required then proceed to glycerol step below. BLEACHING IS NOT ADVISABLE WITH FLOURESCENT ANTIBODIES (for obvious reasons, although I have never tried it so it may be OK)
- Transfer to 75% glycerol (through 25% and 50% glycerol/PBS). Store at 4°C until imaging.
Troubleshooting
Troubleshooting
Methanol
Some antibodies are particularly sensitive to MeOH. Try omitting the dehydration step.
Detergent
Some antibodies are particularly sensitive to detergent. Try swapping PBSTr for PBS.
Antigen retrieval
Although fixation is essential for preserving tissue morphology, this process can also reduce the detectability of proteins by IHC, due to the formation of chemical modifications. Antigen retrieval is an approach to reduce or eliminate chemical modifications. This step should occur just before PK treatment (after rehydration/washing).
Protocol for antigen-retrieval from https://www.aveslabs.com/blogs/protocols/citrate-buffer-antigen-retrieval-protocol