Preparation of Embryos:
1. Fix embryos in 4% PFA
a. Younger than 24hpf fix at 4C O/N
b. Older than 24hfp fix at RT O/N
2. Wash embryos 4X for 20’ with PBST or MABT
3. Treat embryos with molecular grade Proteinase K (1ul in 1 ml)
Proteinase K Treatment
Post Fixation
a. Less 24hpf
no need to treat;
-------b. 24hpf-35hpf
1-2 min
20 min
c. 48hpf
20 min
1 hr
d. 72hpf
30 min
1.5 hr
e. 4-5dpf
45 min
2 hr
4. Stop reaction by immediately post-fixing with 4% PFA at RT
5. Wash embryos 4X for 20’ with PBST; varies depending on time of post fixation.
6. Pre-hybridize embryos in 300ul of Hyb+ (100% Hyb Soln. +torula RNA+heparin) and incubate
O/N in 65C water bath or a minimum length of incubation in Hyb+ buffer is 2 hrs.
7. Embryos can be stored in Hyb+ buffer for a couple of months but the intensity of signals will
progressively decrease.
In Situ Protocol:
1. Hybridization:
a. Add probe into Hyb+ buffer (usually 1:100 is sufficient).
b. Add probe/hyb+ mixture into embryos and incubate in 65-70 C water bath O/N.
2. Washing:
a. Pre-warm the following solutions in a 65-70 C water bath for 20 minutes before use:
100% Hyb soln., 75% Hyb soln., 50% Hyb soln., 25% Hyb soln., 2X SSC, and 0.2X
SSC.
b. Remove probe/hyb+ mixture and transfer to new microcentrifuge tube for future re-use
(2-3). Store at -20 C.
c. Perform the following washes in 65-70 C water bath:
i. 100% Hyb. Soln.
15 min
ii. 75% Hyb. Soln.
15 min
iii. 50% Hyb. Soln.
15 min
iv. 25% Hyb. Soln.
15 min
v. 2X SSC
15 min
vi. 0.2X SSC
2X 30 min
3. Blocking and incubation with pre-absorbed anti-DIG-AP
a. Wash embryos with MAB 2X for 5 mins on the nutator at RT
b. Add 200ul 5X blocking reagent (Roche) + 800ul MAB to embryos and incubate them for
2 hr at RT; can also incubate O/N at 4 C.
c. Remove blocking solution and add 1ml anti-DIG-AP (1:2000; Ab preabsorbed to reduce
background signal in embryos, see antibody absorption for detail) and incubate on nutator
at 4 C O/N.
4. Washing and Staining
a. Save the anti-DIG-AP solution to re-use (6-10 times only).
b. Wash embryos with MAB 4X for 20 min on the nutator at RT
c. For development of anti-DIG-AP staining use staining solution pH9.5; for anti-Fluor-AP
staining, use staining solution pH8.2.
d. Add 1 ml staining solution (pH9.5), incubate 2X for 5 min on nutator at RT (can allow
prolong incubation during lunch break).
e. For every 1ml staining solution (pH9.5), add 4.5 ul NBT and 3.5 ul BCIP (substrates for
AP). Incubate reaction in the dark until blue-black staining is observed at RT or O/N.
Check on color development every 30-1hr when at RT. Change staining solution if no
longer yellow.
Post in situ Immunohistochemistry:
(To perform whole-mount immuohistochemistry on WISH embryos)
Extensive washing to remove anti-DIG and 1 Ab incubation:
All washing steps to be performed on a nutator.
1. After getting an appropriate stained embryos with little background, wash away the staining
solution with PBST, 5min X 2 at RT.
2. Strip anti-DIG-AP by 30min X 2 glycine wash (100mM; pH2.2)
3. Wash excessively to re-equilibrate embryos to neutral pH, 5min for 3X with PBST followed by
washing 4X for 20 mins each with PBST.
4. Block with 1x Roche blocking reagent for at least 1hr at RT
5. Add primary anti-acetylated tubulin (AT) (mouse) and incubate O/N at 4°C.
Washing and incubation with 2 Ab (Anti-mouse HRP or anti-mouse red fluo 594nm):
1. After primary antibody incubation O/N at 4°C, wash embryos with 4 x 20min MAB buffer.
2. Add 1ml of secondary antibody (see below preabsorbed anti-mouse-HRP) to embryos and
incubate them O/N at 4 C on a nutator.
Preparation of preabsorbed Abs:
1. Fix 50 embryos of any stage (or 1-week old) in 4% PFA (in 2ml microcentrifuge tube)
2. Wash embryos with MAB/ PBST for 5X for 10-20min at RT
3. Prepare 400μl 5X blocking reagent (Roche) + 1.6ml MAB. Add to the embryos and block them
for 20 min on nutator at RT.
4. Add 25μl of secondary anti-mouse-HRP Ab (Biorad) to the existing 2ml blocking solution and
incubate for 1hr on nutator at RT.
5. Transfer the 2ml mixture (Blocking reagent + anti-mouse-HRP + MAB) to 15ml Falcon tube.
Top up with 2.4ml MAB, 600μl blocking reagent and 50μl sodium azide (to prevent bacterial
growth); total volume should be 5mls
Washing away 2 Ab:
1. Wash embryos with 4X 20 min MAB buffer at RT on nutator
Preparation for DAB reaction:
1.
2.
3.
4.
5.
Change MAB buffer to H2O.
Dissolve 1 Sigma DAB tablet in 4ml H2O. (Sigma’s 1 tablet in 5ml H2O)
Clarify solution by spinning at max. speed for 5min.
Add 2µl H2O2 for every 1ml DAB solution.
Place embryos on spot glass plate under the dissecting microscope. Carefully dry the embryos
with tissue.
6. Add DAB solution to the embryos and observe them under the microscope while allowing the
reaction to take place.
7. Once an appropriate staining is obtained, stop the reaction by immersing the embryos in fresh
H2O.
8. Add 4% PFA to stop the reaction completely.
In Situ Solutions:
1. 4% PFA: dissolving 40 g of paraformaldehyde (Sigma Cat#158127) in 1L DPBS, and
divided into 50 ml per tube, store at -20C for long-term. For short-term (<2 weeks), store
at 4C
2. Malic acid buffer :
a. 25 ml 1M Malic Acid,
(MAB)
b. 7.5 ml 5M NaCl
c. add SDW to ~200ml
d. adjust pH to 7.5
e. add to ~250 ml
3. PBST : 2ml 20% tween in 400 ml DPBS
4. Proteinase K (1 ul of proteinase K stock (Roche Cat# 03115844001) in 1 ml PBST
5. Hyp+
a. 50 ml 100% Hybridization solution
b. 0.5 ml tRNA
c. 0.5 ml heparin
6. Antibody pre-absorption
Add 20-30 embryos (any stage) to 200ul 5X blocking reagent (Roche) and 800 ul MAB
Incubate at 4C O/N
7. 100% Hybridization Solution (50ml total Vol.)
a.
b.
c.
d.
Formamide
20X SSC
SDW
Tween-20 (100%)
25 ml
12.5 ml
12.5 ml
50 ul
8. 75% Hybridization Solution
a. 100% Hyb Soln
b. 20X SSC
c. SDW
d. Tween-20 (100%)
37.5 ml
3.125 ml
11.875 ml
12.5 ul
9. 50% Hybridization Solution
a. 100% Hyb Soln
b. 20X SSC
c. SDW
d. Tween-20 (100%)
25 ml
6.25 ml
18.75 ml
25 ul
10. 25% Hybridization Buffer
a.
b.
c.
d.
100% Hyb Soln
20X SSC
SDW
Tween-20 (100%)
12.5 ml
9.375 ml
28.125 ml
37.5 ul
11. Staining Buffer
100 ml
50ml
25 ml
a. 1M Tris pH 9.5
10 ml
5 ml
2.5 ml
b. 1M MgCl2
5 ml
2.5 ml
1.25 ml
c. 5M NaCl
2 ml
1 ml
0.5 ml
d. Tween-20 (100%)
100 ul
50 ul
25 ul
e. NOTE: for 50 mls Add 1M Tris pH9.5 and 5 M NaCl and top up to 45 ml with water;
then add 1M MgCl2 followed by top up to 50 mls; correct order will prevent precipitation
of solution!!)
12. 20X SSC
a. 3M NaCl (125g/L)
b. 0.3M Na3citrate-2H20 (88g/L)
c. Adujst pH to 7 with 1M HCl
13. Pre-absorption of antibody
Add 2 ul of anti-Dig-AP (Roche Cat# ) in 200ul 5X blocking reagent (Roche) and 800 ul MAB,
mix with about 30 embryos, and incubate on nuator at 4C O/N.
14. NTB and BICP
Malic Acid Buffer (MAB)