Immunohistochemistry on cells
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PC12 cells were plated on a 96-well plate and differentiated for 3 days with NGF
fixation of the cells with 4 % paraformaldehyde in PBS for 10 min at RT
cells were dried over night at RT
2x washes with PBS 5 min each
permeabilization of the cells for 4 min on ice with permeabilization buffer (20 mM
HEPES pH 7.4, 300 mM sucrose, 50 mM NaCl, 3 mM MgCl2, 0.5 % Triton X-100)
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2x washes with PBS 5 min each
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15 min 1 % H2O2 in methanol
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2x washes with PBS 5 min each
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blocking step: 30 min 5 % FCS in PBS (blocking solution)
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1. antibody (the Anti I4 antibody was diluted 1:50) diluted in blocking buffer for 2.5
h
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3 x 5 min washes with 5 % FCS, 1 % Triton in PBS
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2. antibody (anti-rabbit coupled to HRP) in dilution of 1:1000 to 1:10000 in
blocking solution for30-45 min
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4 x washes with blocking solution containing 1 % Triton for 5 min each
Detection of Antibodies:
For quantification of the bound antibodies the activity of the HRP was checked with a soluble
substrate of HRP (OPD = 0-phenylendediamine, tablets from Sigma).
OPD tablets were dilutet in Phosphate-citrate buffer (capsules from Sigma) and 100 l was
added to each well. The kinetics of the reaction was read at 450 nm every 30 s for 5 min.
Measurement of the cell number/well:
For correcting the results for the cell number in each cells two different methods are possible.
They are both working in the range of 1000-50000 cells/well.
crystal violet staining:
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add 100 l o f 0.1 % crystal violett (Sigma) in Mes Buffer
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shake 20 min at RT
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wash extensively with DDW
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dry the wells in the air
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dissolve the bound colour with 100 l of 10 % glacial acid or 0.4 % SDS
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measure extinction at 590 nm
DAPI:
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add 100 l of 1g/ml DAPI solution to each well
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wash with water
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read the fluorescence at 360 nm (excitation) and 465 nm (emission)
To quantify the number of cells make a standard curve with cells of known number on the
same well as your experiment