斑马鱼免疫荧光

Wholemount Immunohistochemistry of 0- to 40-h Zebrafish Embryos

1

Fixation

：

Fix the embryos in

4% paraformaldehyde

for 3 h at room temperature or overnight

at 4°

C. 20 embryos in each 1.5 mL volume microcentrifuge tube. After fixation, replace the

solution with an equivalent volume of

PBS

(

此步骤后胚胎可存放数周，

但下次用时要

wash

5 × 5 min with PBS).

2

Blocking: Dilute the

goat serum

to 10% (v/v) in

PBT

and incubate 20

–

30 embryos in 0.5 mL of

this solution for 1 h at room temperature on a rocking table.

3

Primary antibody incubation: Remove the blocking solution with a pipet. Dilute the primary

antibody in

PBT plus 1% goat serum

to the appropriate concentration, as recommended by

the supplier. Apply the primary antibody in a minimum

volume of 100 μL

. overnight at 4°

C on

a rocking table with gentle agitation.

4

Washing:

remove

as

much

of

the

primary

antibody

as

possible

and

replace

with

a

large

volume of washing solution (PBT). Change the wash three to five times, with at least 15 min

for each wash with gentle rocking at room temperature (

洗的时间长，背景会降低

).

5

Dilute the secondary antibody

：

Dilute the secondary antibody in

PBT plus 1% goat serum

to

the

appropriate

concentration,

as

recommended

by

the

supplier.

overnight

at

4°

C

on

a

rocking table with gentle agitation. keep the embryos in the dark from this step on to prevent

bleaching.

6

Remove the secondary antibody and wash the embryos in PBT. Change the wash three to five

times, with at least 15 min for each wash with gentle rocking at room temperature. Finally

transfer the embryos to PBS alone prior to the detection steps. At this point, if fluorescence is

used,

clear

the

embryos

in

graded

glycerols

and

mount

in

70%

glycerol

containing

an

antibleaching agent such as

Citifluor(

抗荧光衰退剂

)

(Agar Scientific Ltd).

7

Examine use confocal microscope.

4%

多聚甲醛

-0.1mol/L

磷酸缓冲液（

pH7.3

）

:

配制方法：称取

10g

多聚甲醛，置于三角烧瓶中，加入

125

～

200ml

0.1mol/L

磷 酸缓冲

液（

Phosphate

Bu ffer

以下简称

PB

）

，加热至

60

℃左右，持续搅拌（或磁力搅拌）使粉

末完全溶解，通常需滴加少许

1n

N aOH

才能使溶液清亮，最后补足

0.1mol/L

PB

于

250ml

，充分混匀。

PBS:

8.0 g NaCl, 0.2 g KCl, 1.44 g Na

2

HPO4, 0.24 g KH

2

PO4 in 1 L dH2O, pH 7.4,

PBT:

PBS plus 0.8% Triton X-100 (Sigma).

goat serum (sigma)

graded glycerols:

30%, 50%, and 70% glycerol in PBS

Citifluor(

抗荧光衰退剂

) (Agar Scientific Ltd)