Immunofluorescence on Cultured Cells (a double staining protocol)
1. Plate cells in chambered slides.
2. Culture 24-72 hours.
3.
Wash with PBS (don't dry the cells).
4. Fix cells by one of the following methods:
1% formalin in PBS for
10 minutes
80% methanol in PBS for 10 minutes
Cold acetone for 5 minutes, air dry
4% paraformaldehyde for 10 minutes.
5.
Permeabilize the cells with 0.1% Triton X-100 in PBS for 5 minutes at room temperature.
6. Block non-specific antigen with 10%
normal serum for 20 minutes at room temperature.
7. Incubate with primary antibody at proper dilution (1:100,for example) in TBS for
1 hour at
room temperature ( or 45 minutes at 37 centigrade, or 4 centigrade over night).
8. Wash with 0.1% Triton
X-100 in PBS 3x5 minutes, at room temperature.
9. Incubate with fluorescence (FITC-, for example) conjugated secondary antibody
at proper dilution
(1:100, for example) in TBS for 45 minutes at room temperature.
10.Wash with 0.1% Triton X-100
in PBS 3x5 minutes, at room temperature.
11. Repeat Step 7 with another primary antibody.
12. Repeat Step 8.
13. Repeat Step
9 with another fluorescence (Rhodamine-, for example) conjugated secondary antibody.
14. Repeat Step 10.
15. Mount the slides with antifade
solution with DAPI (for counterstaining).
16. Seal the slides with nail polish.
17. Observe under microscopy, or keep at -20 centigrade.
Immunoflrorescence
on Paraffin Sections
1. Preparing the slides
-Heat the slides at 60 centigrade for 60 minutes.
-Rinse in toluene twice, 10 minutes
each.
-Hydrate in ethanol in gradiently decreased concentrations.
100% ethanol 1x5 minutes
90% ethanol 1x5 minutes
70% ethanol 1x5 minutes
water 1x5 minutes
2. Antigen Retrieval
-Boil slides
in 1:100 diluted unmasking solution (Vector Laboratories #H-3300) in a
microwave oven set for 3 cycles: P5(power level), time
4 minutes for heating, time 1
minute for pause.
-Cool down at room temperature.
3. Permeabilize sections
-Incubate in 0.1% Triton
X-100 in TBS for 30 minutes at room temperature in a
humidified chamber.
4. Work with Primary Antibody
-Remove the permeabilizing
buffer.
-Block with 10% normal serum in TBS for 20 minutes.
-Wash with 0.1% Tween-20 in TBS 3x5 minutes.
-Remove the blocking buffer.
-Incubate
with primary antibody in proper dilution (1:100, for example) in blocking buffer,
for 1 hour at room temperature, or overnight at
4 centigrade.
5.Work with Secondary Antibody
-Wash with 0.1% Tween-20 in TBS 3x5 minutes.
-Incubate with fluorescence conjugated
secondary antibody with proper dilution (1:100, for example)
in blocking buffer, for 45 minutes at room temperature.
-Wash
with 0.1% Tween-20 in TBS 3x5 minutes.
6.Mount the slides
-Mount the slides with antifade solution with DAPI.
-Observe the sections
under microscopy, or keep them at -20 centigrade.
Note: The dilution for primary antibody and secondary antibody is changeable
accordingly, so does
it with incubation time.
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Ch 8.Immunohistoch / immunology
Ch 10.GC/MS, NMR and Proteomics