Fixation and Permeabilization
1. Wash
the cover glass seeded with cells in the culture plate with 1× PBS bufferfor
3 minutes and repeat 3 times.
2. Fix
the cultured cells for 15 minutes with 4% paraformaldehyde, and then wash the
cover glass with 1× PBS for 3 minutes and repeat 3 times.
3. Permeate
the cells at room temperature for 15 minutes with 0.5%Triton X-100 (prepared
with 1× PBS) (This step can be omitted for antigens that are expressed on cell
membranes), and then wash the cover glass with 1× PBS for 3 minutes
and repeat 3 times.)
Blocking
4. Blot
up the absorbent paper with 1×PBS,
and add 5% normal serum (Sharing the same or similar species with secondary antibodies)
drop by drop on the cover glass, then incubate it at room temperature for 1
hour.
Antibody incubation
5. Use
absorbent paper to aspirate the blocking solution without washing
the cover glass, and add sufficient and diluted primary antibodies drop by drop
on each cover glass and then put them into wet box to be incubated at 4℃ overnight.
6. Add
fluorophore-conjugated secondary antibodies: firstly wash the cover glass with PBST for 3 minutes and repeat 3 times,and then add the diluted
fluorophore-conjugated secondary antibodies drop by drop after blotting up the
redundant liquid on the cover glass with absorbent paper. Finally put them into
wet box to be incubated at 37℃ for 1
hour, and wash the section with PBST for 3 minutes and repeat 3 times.
Attention: All of the operation steps should be operated in the dark after adding the
fluorophore-conjugated secondary antibodies.
Fixing and taking pictures
7. Nuclear
staining: add DAPI drop by drop and incubate for 5 minutes in the dark, and
then stain the nucleus of the specimen. Finally wash away the redundant DAPI with
PBST for 5 minutes and repeat 4 times.
8. Blot up the
liquid on the cover glass with absorbent paper, then use antifade mountant to
mount the cover glass, finally observe and collect images under a fluorescence
microscope.
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