In this section:
- Dent's Fix (Methanol/DMSO)
- Bleaching solution (Mayor et al. 1995)
- Goat Serum (GS)
- Secondary Antibodies Coupled to Horseradish Peroxidase
- Murray's Clearing Medium
MEMFA (MOPS/EGTA/Magnesium Sulfate/Formaldehyde Buffer)
|1X||10X||for 1 L 10X|
|MOPS (pH 7.4)||0.1 M||1.0 M||209.27 g|
|EGTA||2 mM||20 mM||7.608 g|
|MgSO4||1 mM||10 mM||2.46 g|
- Prepare a 10 X solution without the formaldehyde for storage. Add fresh formaldehyde(1/10 volume of standard 30% stock) prior to use. The solution will turn yellow with age and when autoclaved.
Dent's Fix (Methanol/DMSO)
- 4 volumes methanol
- 1 volume DMSO
Bleaching solution (Mayor et al. 1995)
1% H202 5% formamide 0.5 X SSC(standard saline citrate)
20x PBS (phosphate-buffered saline)
- Na2PO4 H20 - 5.12 g
"Ha2HPO4 - 23.88 g
- NaCl - 204.4 g
- distilled water - 800 ml
Adjust pH to 7.4 and volume to 1 liter.
Goat Serum (GS) (Life Technologies; Mycoplasma-tested) 500 ml
Heat deactivate complement for 30 minutes at 56 deg C. Store as 1 ml aliquitest at -20 deg C.
- PBS (phosphate buffered saline)
- 2 mg/ml BSA (bovine serum albumin) (Fraction 5, reagent grade)
- 0.01% Triton X-100
DAB (Diaminobenzidine; Polyscience)
- 1 mg/ml in PBT
- Filter through a 0.2-um flitter and store as 1 ml aliquots at -70 deg C.
H2O2 (Hydrogen peroxide; Sigma 30% stock solution)
- Store at 4 deg C. Dilute 1/2x in PBT just before use.
Secondary Antibodies Coupled to Horseradish Peroxidase (e.g. Bio-Rad)
- Store as 100 ul aliquots at -20 deg C.
Murray's Clearing Medium (Benzyl Benzuate/Benzyl Alcohol, BB/BA)
- 2 volumes of benzyl benzoate
- 1 volume of benzyl alcohol