PROTOCOL 012: ZEBRAFISH WHOLE MOUNT IMMUNOHISTOCHEMISTRY

 

REAGENTS

 

Fixation:

 

       4%PFA

       Methanol

       1 microM 1-phenyl 2-thiourea (PTU) (if appropriate)

       PBST (1X PBS/0.1% Tween-20)

 

Immunohistochemistry (IHC):

 

       Ice-cold acetone

       DAPI

       PBST

       Block (PBST + 5% Sheep Serum)

       Primary antibody

       Secondary antibody

       Rehydration Solutions:

95% MeOH + 5% PBST

75% MeOH + 25% PBST

50% MeOH + 50% PBST

25% MeOH + 75% PBST

 

PROCEDURE

 

A.    Fixation:

 

1.     If embryos are to be fixed later than 24hpf, rear them in PTU to prevent pigmentation (unless this interferes inherently with your experiment). PTU should be added to E2 medium prior to initial onset of pigmentation (28 somites).

 

2.     Dechorionate embryos

 

a.     24 hpf: dechorionate after fixation

b.     >24 hpf: dechorionate prior to fixing in PFA

 

3.     Fix embryos in 4% PFA at time-points of interest. Place approximately 15 embryos/ml 4% PFA in a microfuge tube and rock 3-4 hours at room temperature. Alternatively, embryos can be incubated overnight with rocking at 4C.

 

4.     Rinse embryos 1X in 1 ml PBST at RT. Remove PBST.

 

5.     Add 1 ml methanol, invert to mix, and allow embryos to settle to the bottom. Remove methanol and replace with fresh methanol.

 

6.     Store in methanol at -20C until ready to use.

 

B.    IHC:

 

1.     Rehydrate embryos in gradual MeOH/PBST progression:

 

95% MeOH + 5% PBST

75% MeOH + 25% PBST

50% MeOH + 50% PBST

25% MeOH + 75% PBST

 

2.     Wash at least 4 X 5 minutes in 1ml PBST

 

3.     Permeabilize embryos in 1ml ice-cold acetone for 8 minutes at -20C

 

4.     Wash at least 4 X 5 minutes in 1ml PBST-20%

 

5.     Incubate the embryos twice for 1 hour in 1ml block

 

6.     Add primary antibody (recommended 1:100 concentration for first time application)

 

7.     Incubate overnight with gentle rocking at 4C

 

8.     Wash embryos 5 X 10 minutes in 1ml block at RT

 

9.     Wash 3 X 10 minutes in 1ml PBST

 

10.  Add appropriate 2 antibody (recommended 1:750 concentration)

 

11.  Wrap in foil and incubate over night at 4C with gentle rocking

 

12.  Wash embryos 5 X 10 minutes in 1ml block

 

13.  Wash 3 X 10 minutes in 1ml PBS 0.1% Tween

 

14.  Apply optional fluorescent stains (e.g. DAPI at 1:50 dilution) X 30 minutes

 

15.  Remove DAPI, add 1ml block and store at 4C in the dark until analysis

 

16.  Mount and view embryos.