Immunohistochemistry

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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