IMMUNOHYSTOCHEMISTRY
The protocol has been optimised for the detection of the fibronectin
isoform containing the Extra Domain B and then extensively used both for
the detection of other known antigens and the characterisation of antibodies
directed against unknown antigens.
As primary antibody, we use recombinant antibody fragments in the scFv
format, isolated from the ETH-2 library. Both purified scFvs and
antibody fragments released in bacteria supernatant can be used as primary
reagents.
Since the scFvs from the ETH-2 library have a FLAG sequence at the
C-terminus, commercially available anti FLAG M2 antibody is used.
The sequence of reagent application is: primary antibody together with
anti-FLAG antibody, biotinylated secondary antibody (Biotinylated goat
anti-mouse IgG), preformed avidin-biotin-enzyme complex (ABC), substrate
solution.
REAGENTS
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Streptavidin-Biotinylated Alkaline Phosphatase Complex BIOSPA F014-62
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Biotinylated Goat anti-mouse IgG BIOSPA M001-62
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Lavamisole Sigma L9756
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Fast Red TRSalt Sigma F8764
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Naphtol AS-MX Phosphate (Free acid) Sigma N4875
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Glycergel mounting Medium Dako 0563
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Gill Hematoxilin Solution n.2 Sigma HHS-16
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Aprotinin Sigma A6279
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N,N dimethylformammide
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Tween 20
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Fetal Calf Serum
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MgCl2
BUFFERS
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TBS:
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50mM TRIS
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100 mM NaCl
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Sol. A:
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TBS pH 7.4
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0.05% Tween 20
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3% BSA
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Sol. Fast Red:
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TRIS-HCl 0.1M pH 8.2
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58.8 ml
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N,N-DimethylFormammide
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1.2 ml
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Naftolo ASMX Phosphate
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12 mg
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Levamisole sol.
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60 µl
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The solution can be stored at 4°C for 7-14 days. In this solution, Fast Red has to be dissolved only just before its use.
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Levamisole sol. (1M sol.):
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TTRIS-HCl 0.1M pH 8.2
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1 ml
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Levamisole powder
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240.8 mg
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MgCl2 sol. (stock solution 100x):
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0.2 M MgCl2 in TBS
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This solution can be stored at -25°C for a long time
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Fast Red Substrate:
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Red powder must be added to the Fast red solution just before
its use
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Add for example 5 mg FastRed powder to 5 ml Fast Red solution
Filter 0.45 µ
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Glycergel:
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Warm at 50°C
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While mounting your slides, leave it upside-down in warm.
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PROTOCOL
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Fix the sections in cold aceton (-20°C)
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10'
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Let the slides dry at RT
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30'
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Immerse in Sol. A
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5'-10'
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dry with paper without touching the sections
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Block with FCS/Sol.A (1/5) in a moist chamber
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30'
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Let the solution fall down into paper
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Add the primary antibodies (es. scFv and M2 Ab) diluted in Sol.B
(100 µl/section)
Incubate in a moist chamber
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1h
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NOTE:
-The final concentration of a scFv should be in the range of 2-30 µg/ml
Make dilution series of the primary antibody to calculate the optimal
concentration that you need
-Together with the antibody fragment recognising the antigen of interest,
add the anti FLAG antibody M2. The final concentration of the M2 antibody
should be in the range of 10-50 µg/ml. Try with a dilution series to find
the optimal working concentration for your protocol.
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Rinse twice by directly applying the buffer on the section with a Pasteur
pipet, followed by immersion of the slides on buffer twice, 5' each time.
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Incubate in a moist chamber
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30'
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Make a dilution series of the antibody in the range of 1/100-1/400
to choose the optimal concentration of the secondary antibody for your
protocol.
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Wash by immersion in Sol.A
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5'+5'
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Dry with paper the back of the slides
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Add the Streptavidin-Biotinylated Alkaline Phosphatase Complex.
Dilute the complex in Sol. B and add MgCl2 (final concentration 0.002M).
Usually a dilution 1/400 of the complex is necessary.
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Incubate in a moist chamber
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30 '
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Wash by immersion in Sol.A
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5'+5'
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Dry with paper the back of the slides
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Add the substrate (dissolve fastRed powder in Sol. Fast Red as already explained in the "buffer" section). Use 500 ul/slide.
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Incubate in a moist chamber
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20 '
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Wash with deionised water: twice directly applying the water on
the section with a Pasteur pipet and then leave the slides in water.
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Transfer the slides in Gillís Hematoxilin solution n.2 for
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50''
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Quickly transfer the slides in water and rinse with water for 6
times
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Mount the slides with Glycergel
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