Immunohistochemical staining of zebrafish embryos(V.Korzh, IMCB, NUS, e-mail vlad@imcb.a-star.edu.sg)
There is no perfect protocol for all applications. Any protocol you'll get needs to be
optimized to conditions in the lab by varying conditions of staining. The protocol
below represents a compilation of many years of experience, but it is by no means a
"perfect" protocol. As new reagents arrive on a market continuously it might be a
better fixative or antibody available. So it is up to you to get a perfect staining by
changing concentration of antibody or increasing washing time.
Whole mount protocol
- dechorionate embryos (optionally late embryos can be anaesthetize by dissolving a
few crystals of MS222 in PB or by keeping embtyos for 30’ on ice prior to fixation
and use ice-cold fixative, so they will not curl)
- fix 3 h at room temperature (RT) or overnight (ON) at 4oC in 2-4% PFA in PBS. (To
preserve tissue and reveal fine processes, one can use fixation with 4%PFA-4%
sucrose ON at 4oC).
Some antibodies are able to detect antigenes only after fixation with 3.7% formalin or
Dent's fixative or Histochoice (for recipes of fixatives see below).
- wash several times in PBS (eg. 4x10 min)
- (optionally embryos can be preserved for few month in MeOH in -20oC, in this case
before starting staining change from MeOH to PBS in 2-3 steps. But remember that
best results are seen with freshly fixed material)
- to reduce background peroxidase staining embryo must be treated with MeOH, 10%
H2O2 for 1 h +4oC, and transferred to MeOH for storage. This is a necessary step for
embryos of older stages, when peroxidase in blood can cause a substantial
background. From here embryos could be processed for sectioning and fluorescent
staining on sections.
- to improve penetration of antibodies embryos can be treated to remove lipids from
the cell membrane. Embryos should be washed in deionized water or MQ water for 10
sec(to remove salts) and immediately cold aceton must be added (-20oC) and embryos
put into -20oC (freezer) for 8 min or longer depending on stage. After this treatment
aceton removed, deionised water added (to remove traces of aceton) and PBS washes
3 x 10 min made
- block nonspecific binding sites - incubate at least 1 h RT in 10% serum in PBT or
TBST
- incubate in primary antibody, diluted in PBT + 1-10% serum, ON +4o
For double labeling combine different primary antibodies (eg. rabbit polyclonal
(Islet-1)+ mouse monoclonal (HNK-1) at proper concentration + 1-10% serum.
- remove primary antibodies and save (can be reused 2-3 times) with Na azid + EDTA
- wash embryos several times with PBT - rinse embryos briefly 2-3 times and 3x3040’ in addition
- apply secondary antibodies, diluted in PBT + up to 10% serum and incubate 2-3 h
RT or ON 4oC
- wash embryos several times with PBT - rinse embryos briefly 2-3 times and 3x3040’ in addition
- if using ABC kit prepare a mixture of solutions A and B (0.5 ml PBT add 10 l A ,
vortex+ 10 l B, vortex, leave for 30 min at RT, dilute up to 6 times with PBT, add
serum up to 10% and finally apply to embryos for 2 h
- if using secondary antibodies directly conjugated to POD or AP or fluorophore, the
last step must be omitted
- wash embryos several times with PBT - rinse embryos briefly 2-3 times and 3x3040’ additionally (omit this stage when trying to detect fine cellular processes by
fluorescent staining),
- wash embryos with PBS 2x 5-10’ (to remove PBT, which may inhibit POD reaction)
and transfer them to the well of 24-well plates
- carefully prepare DAB solution by dissolving DAB in 0.5xPBS (0.5-1.0mg/ml),
- filter through disposable filter (optional)
(DAB is presumably potential carcinogen, handle this solution carefully using
gloves)
- add 1 ml of DAB solution to embryos, incubate for 30’ at RT or on ice in dark
(seems to reduce background) (the remaining DAB solution can be stored at -20oC
indefinitely and thawed before usage)
- start staining reaction, adding 0.33l of 30% H2O2
- control staining reaction under dissecting microscope
- after reaching proper level of staining, stop reaction by changing staining solution to
MeOH (remember that a level of staining sufficient for observation of embryos as
whole mount usually is too weak for sectioning. Thus, embryos should be overstained
as whole mount for getting good images on sections. Alternatively stain sections
directly).
- save embryos in MeOH or rehydrate them slowly. At that stage they can be destained
in PBS +4o ON
- transfer embryos step by step to 50% glycerol-PBS
Mounting
- for whole mounts side view mount between large coverslips using 2-3 small cover
slips as spacers sealed by DPX, Permount or other mounting media
- for younger stages and to get high resolution pictures prepare the flat specimen by
scratching yolk away with sharp needles in a spotted glass plate (yolk granules will
give reflections). Place an embryo without yolk on a microscopic slide, remove excess
of PBS-glycerol, cover with a smallest fragment of a coverslip (big coverslip will
squash an embryo, orient properly by moving the coverslip fragment and fill space
under the coverslip with PBS-glycerol.
In general it is more convenient to view various parts of older embryos from different
angles - the head from the top and the tail from the side. For that embryos must be
divided at the level of anterior spinal cord in two pieces. Mount these parts separately
correspondingly.
Seal edges of coverslip with colorless nail polish.
All staining and washing must be done on slow speed rotary shaker (Nutator) in
Eppendorf tubes or plates with 24 wells (2 ml) for tissue cultures.
Appendix
Concentrations of some antibodies (may not be true for the particular batch you are
working with).
Primary antibody
Islet - 1 (rabbit IgG, polyclonal) - 1:500
Islet - 1 (mouse IgG, Mab)
1:5
3A10 (mouse IgG, Mab)
1:3
Fret 43 (mouse IgG, Mab)
1:4
En2 En (rabbit IgG, polyclonal) 1:100
En1 4D9 (mouse IgG, Mab)
1:5
MZ15
(mouse IgG, Mab)
1:300
integrin (rabbit IgG, polyclonal) 1:250
GABA (guinea pig)
1:500-2000
neurofilament 200 (Sigma) (rabbit) 1:100
anti-N-CAM (Sigma)
1:100
b-catenin (mouse, IgG)
1:250-500
neuromodulin (GAP-43)(mouse, IgG) 1:100
bNOS (rabbit, IgG)
1:250
acetylated tubulin (mouse, IgG)
1:200
anti-HNK-1/N-CAM (mouse IgM) 1:400
F59 (mouse IgG)
1:10
Secondary antibodies
Secondary biotinilated antibodies anti - rabbit IgG and mouse IgG &IgM - 1:500
Secondary HRP tagged anti-rabbit IgG and mouse IgG&IgM
1:250
Fluorescent tagged (for staining of sections)
TRITC labeled secondary anti-rabbit
Streptavidin-FITC
1:100
1: 200
Film exposure (manual for fluorescence)
red (TRITC, RITC) - 4, 8, 16 sec
green (FITC) - 1, 2, 4 sec
Fixation:
1) 2-4% PFA (2% PFA to be used only for very young embryos followed by 4% PFA
fixation, for most applications 4%PFA should be used)
warm PFA in PBS on a heater/stirrer at 70oC until dissolves completely, cool,
store at 4oC and use no longer than 1 month
2) MOPS-based formalin 3.7% formalin (use commercial 37% solution as a stock)
0.1M MOPS, pH 7.4
1 mM MgSO4
2mM EGTA
3) Dent's fixative: 20%DMSO/80% EtOH
4) Histochoice (Amresco). Probably this is the same or similar to Dent's.
-gal staining solution:
1 ml
10 ml
0. 922 ml
10 ul
10 ul
8 ul
1 mg (50 ul)
9.22 ml
100 ul
100 ul
80 ul
10 mg (500 ul)
PBS
K3Fe(CN)6(0.5M)
K4Fe(CN)6 x 3H2O (0.5M)
MgCl2 (0.25M)
X - gal (20mg/ml stock solution)
This reaction takes place at 37oC and develops slowly. It may take up to two days.
You can transfer embryos to +4oC overnight and continue next day. Pls, monitor
progression of reaction under dissecting scope from time to time. When using in
combination with POD staining, stain for -gal first followed by POD staining at the
end.