General Protocol for Immunohistochemistry

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The procedure for whole mount immunohistochemistry in X. laevis is identical to the protocol for X. tropicalis (see Sive et al 2000). We and others have found antibodies that detect specific antigens in X. laevis do so equally well in X. tropicalis.

Please see our paper for more details:

Khokha et al. 2002

See alternate protocol for Acetylated Tubulin staining, adapted from a protocol provided by Basudha Basu. It works well for epidermal cilia.

Thanks to Nancy Hoo for creating this page.

Antibody Stain	1° Antibody	Cat #	Dilution	2° Antibody	Cat #	Dilution
12-101 Muscle Cells	12-101	_	Undiluted	Peroxidase Conjugated Affinipure Goat Anti-Mouse IgG (H+L)	"Jackson" 115-035-003	1:500
Phospho-Histone H3 Mitotic Cells	Anti-Phospho-Histone H3	"Upstate" 06-570	1:1000	Goat Anti-Rabbit IgG (H+L) Conjugate	"Bio-Rad" 172-1019	1:1000
Prox-1 Lymphatic Cells	Polyclonal Rabbit Ag	"Upstate" 145-930-002	1:1000	Goat Anti-Rabbit IgG (H+L) Conjugate	"Bio-Rad" 172-1019	1:1000
Tor 70 Notochord	Tor 70	_	1:5	Goat Anti-Mouse IgM	"Jackson" 115-035-020	1:1000

Fixation of embryos:

Reagents:

MEMFA
10% 10X MEMFA salt
10% formaldehyde
80% water
Methanol

1) Fix embryos in MEMFA for 1-2 hr

2) Dehydrate in several MeOH washes

3) Embryos can now be stored long term at -20°C

BEGIN Antibody Stain DAY 1:

Reagents:

Methanol
PBT
1X PBS
2 mg/mL BSA
0.1% Triton X-100
1º Antibody

Rehydration:

5 minutes each wash

1) ___ MeOH
2) ___ 75% MeOH 25% H2O
3) ___ 50% MeOH 50% H2O
4) ___ 25% MeOH 75% H2O
5) ___ 100% PBT

Blocking: (optional for 12/101)

1) ___ 5 min in PBT
2) ___ 5 min in PBT
3) ___ 1 hr in PBT

Primary Antibody:

___ 1º Antibody 4 hr RT or 4°C O/N

DAY 2:

Reagents:

PBT
2° Antibody

PBT Washes:

1) ___ 10 seconds
2) ___ 20 min
3) ___ 20 min
4) ___ 20 min
5) ___ 20 min
6) ___ 20 min

Secondary Antibody:

1) ___ 2° Antibody 4 hr RT or 4°C O/N

DAY 3:

Reagents:

PBT
DAB

PBT+0.5 mg/mL DAB

1000X H2O2 solution
1:1 dilution of 30% H2O2 in PBT
Bleach

0.5X SSC
5% formamide
4% 30% H2O2 stock solution

PBT Washes:

1) ___ 10 seconds
2) ___ 20 min
3) ___ 20 min
4) ___ 20 min
5) ___ 20 min
6) ___ 20 min

DAB Detection:

1) ___ 10 min in DAB at 4°C
2) ___ Add 1X H2O2 solution to initiate reaction
3) Monitor reaction.

Usually takes 5-10 minutes. Reaction may be more optimal at 4°C rather than RT.

END Antibody Staining

1) ___ 5 min in PBT
2) ___ 5 min in PBT
3) ___ For a second stain, start over at 2°
Antibody after two 30 min PBT washes

Bleach:

1) ___ 2 hr in Bleach under fluorescent light
and then O/N at RT under ambient light