General Protocol for Immunohistochemistry
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:
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 | Image | 1° Antibody | Cat # | Dilution | 2° Antibody | Cat # | Dilution |
---|---|---|---|---|---|---|---|
12-101
|
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12-101 |
_ |
Undiluted |
Peroxidase Conjugated Affinipure Goat Anti-Mouse IgG (H+L) |
"Jackson"
|
1:500 |
Phospho-Histone H3
|
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Anti-Phospho-Histone H3 |
"Upstate"
|
1:1000 |
Goat Anti-Rabbit IgG (H+L) Conjugate |
"Bio-Rad"
|
1:1000 |
Prox-1
|
Polyclonal Rabbit Ag |
"Upstate"
|
1:1000 |
Goat Anti-Rabbit IgG (H+L) Conjugate |
"Bio-Rad"
|
1:1000 |
|
Tor 70
|
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Tor 70 |
_ |
1:5 |
Goat Anti-Mouse IgM |
"Jackson"
|
1:1000 |
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
5 minutes each wash
1) ___ MeOH
2) ___ 75% MeOH 25% H2O
3) ___ 50% MeOH 50% H2O
4) ___ 25% MeOH 75% H2O
5) ___ 100% PBT
1) ___ 5 min in PBT
2) ___ 5 min in PBT
3) ___ 1 hr in PBT
___ 1º Antibody 4 hr RT or 4°C O/N
DAY 2:Reagents:
PBT
2° Antibody
1) ___ 10 seconds
2) ___ 20 min
3) ___ 20 min
4) ___ 20 min
5) ___ 20 min
6) ___ 20 min
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
1) ___ 10 seconds
2) ___ 20 min
3) ___ 20 min
4) ___ 20 min
5) ___ 20 min
6) ___ 20 min
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.
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