TITLE: Immunohistochemistry (IHC) Whole-Mount Antibody Staining 
AUTHORS: Talia Pittman

[DESCRIPTION]
Immunohistochemistry (IHC) Whole-Mount Antibody Staining protocol @FishFloorUCL

[STEPS]
SECTION: Introduction
1. Protocol based on Jenny Regan's GFP protocol, modified by Tom Hawkins thomas.hawkins@ucl.ac.uk
SECTION: Fix and dehydrate
2. 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.
SECTION: (Day 1) Rehydrate
3. This step applies to PFA fixed, MeOH stored embryos only

1 x 5mins 50% MeOH / 50% PBTr
3 x 5mins PBTr
SECTION: (Day 1) Permeabilise
4. 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)
SECTION: (Day 1) Block
5. 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.
SECTION: (Day 1) Apply Primary Antibody
6. 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

SECTION: (Day 2) Wash
7. 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.
SECTION: (Day 2) Block Endogenous Peroxidases
8. 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
SECTION: (Day 2) Apply Secondary Antibody
9. 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.
SECTION: (Day 3) Wash
10. 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.
SECTION: (Day 3) DAB Detection
11. 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
SECTION: (Day 3) &#x27;ABC&#x27; Amplification
12. 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).
SECTION: (Day 3) Wash
13. 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.
SECTION: Troubleshooting
14. 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