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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 that 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

Thanks to Nancy Hoo who put this page together.

Antibody Stain Image Primary Antibody Catalog # Dilution Secondary Antibody Catalog # 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

The following protocol can also be downloaded as a PDF or Word.

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) o MeOH

2) o 75% MeOH 25% H2O

3) o 50% MeOH 50% H2O

4) o 25% MeOH 75% H2O

5) o 100% PBT

 

Blocking: (optional for 12/101)

1) o 5 min in PBT

2) o 5 min in PBT

3) o 1 hr in PBT

Primary Antibody:

o 1º Antibody 4 hr RT or 4°C O/N

 

DAY 2:

Reagents:

PBT

2° Antibody

PBT Washes:

1) o 10 seconds

2) o 20 min

3) o 20 min

4) o 20 min

5) o 20 min

6) o 20 min

 

Secondary Antibody:

1) o 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) o 10 seconds

2) o 20 min

3) o 20 min

4) o 20 min

5) o 20 min

6) o 20 min

 

DAB Detection:

1) o 10 min in DAB at 4°C

2) o 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) o 5 min in PBT

2) o 5 min in PBT

3) o For a second stain, start over at 2°

Antibody after two 30 min PBT washes

 

Bleach:

1) o 2 hr in Bleach under fluorescent light

and then O/N at RT under ambient light