Date : 2024-01-18

Author : V Chong-Morrison

Adapted from protocol by A Faro based on the following publication:

Turner, K.J., Bracewell, T.G., Hawkins, T.A. (2014). Anatomical Dissection of Zebrafish Brain Development. In: Sprecher, S. (eds) Brain Development. Methods in Molecular Biology, vol 1082. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-655-9_14

Whole-mount zebrafish immunohistochemistry

Introduction

This protocol has been tested with the following antibodies (antibody, supplier (may be more than one), clone ID (if known)):

• rabbit GFP - Torrey Pines BioLab (TP401)
• chicken GFP - abcam (ab13970)
• mouse Tubulin (Acetyl Lys40) - GeneTex (GTX16292) Sigma (T7451), 6-11B-1
• rabbit RFP (mCherry-suitable) - MBL (PM005)
• rabbit sox10 - GeneTex (GTX128374)

I generally use primary antibodies at 1:200, and secondary Alexa Fluor antibodies at 1:400.

Materials

• 4% Paraformaldehyde (PFA) in 1X Phosphate Buffered Saline (PBS)
• 1X PBS + 0.8% Triton-X100 (also works with Tween-20) i.e. PBST
• Methanol (MeOH)
• Proteinase K (10 mg/ml = 1000X, 20 mg/ml = 2000X)
• Normal Goat Serum
• Dimethylsulfoxide (DMSO)
• Low Melting Point (LMP) agarose
• Glycerol

Procedure

Fixation

Options

• 4% PFA overnight at 4C
• 2% PFA over-weekend at 4C
• 4% PFA 1 hour/24 hpf at RT
• (If post-fixing dissection required) Sweet fix (4% PFA + 4% sucrose) for 3 hrs at RT or overnight at 4C

Rinse and dehydration

1. Rinse 2x in PBST.
2. Wash 3x 10 mins PBST on side on shaker.
3. Dehydrate into MeOH:
   • 1x 5 mins 50% MeOH/50% PBST
   • 2x Rinse in 100% MeOH
4. Store at -20C in 100% MeOH at least overnight up to 6 months.

NOTE Rinsing and dehydrating with plain PBS works too.

Day 1

Rehydration

1. 1x 5 mins 50% MeOH/50% PBST
2. 3x 5 mins PBST

Permeabilisation with Proteinase K in PBST

1. Varies with embryo age. Perform at RT with tube lying on the side on a nutator:
   • Up to tailbud: no PK
   • 2-10ss: in and out of 1X PK
   • 10-15ss: 1 min 1X PK
   • 16-26ss: 2 mins 1X PK
   • 24h: 10-15 mins 1X PK
   • 30h: 20 mins 1X PK
   • 36-48h: 30 mins 1X PK
   • 2.5d: 30 mins 1.5X PK
   • 3d: 30 mins 2X PK
   • 4d: 30 mins 3X PK
   • 5d: 30 mins 4X PK
2. Rinse 3x in PBST.

Post-fixing

1. 4% PFA for 20 mins at RT.
2. Wash 3x 5 mins PBST.

Blocking

1. Make blocking solution fresh each time:
   • 10% normal goat serum (NGS), 1% DMSO in PBST
2. Incubate 1 hour at RT on nutator.

Primary antibodies

1. Incubate in blocking solution at least overnight at 4C on nutator.

Day 2

Washes

1. Remove primary antibody (can be kept at 4C for reuse within a week).
2. Rinse 3x in PBST.
3. Wash 6x 30 mins in PBST on nutator.

NOTE If you incubate primary antibody over several days then wash more.

Secondary antibodies - keep dark

1. Centrifuge secondaries at 500g for 5 mins to pull down precipitates. Use supernatant for staining.
2. Incubate in PBST + secondary antibodies overnight at 4C or 3-4 hours at RT on nutator.

Day 3

Washes and storage/imaging - keep dark

1. Rinse 3x in PBST.
2. Wash 6x 30 mins on nutator.
3. If desired, perform a long over-weekend wash at 4C.

Storage - keep dark

1. Rinse PBST with PBS.
2. Transfer to 50% glycerol/PBS.
3. Mount in 1% LMP agarose, immerse (if required) with PBS.
4. Image as soon as possible, samples keep alright for about a week or so.