Immunohistochemistry

In this section:

MEMFA

Dent's Fix (Methanol/DMSO)

Bleaching solution (Mayor et al. 1995)

Goat Serum (GS)

PBT

DAB

H2O2

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
Formaldehyde	3.7%

• 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.

PBT

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