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Host species: Goat
Conjugation: Alexa Fluor® 488
The image shows lack of colocalization between DAPI staining (pseudocolored green)and LEF-1 immunostaining with this Ab (pseudocolored red). Section of bell-stage embryonic tooh showing dental papilla to the right and enamel organ to the left.
Image of LEF-1 antibody is pseudocolored. We did it in order to
maximize the visual appereance of colocalization with DAPI nuclear staining,
if applicable. DAPI should go on the blue channel (green in the image).
LEF-1 should go on the green channel (red in the image).
We tried the three fixatives: Paraformaldehyde, acetone and methanol.
Regarding paraformaldehyde itself, we have a broad experience using it as a
fixative to detect both cytoplasmic and nuclear proteins in mouse frozen
sections (IHC-Fr), and never had a problem with it. I must say that we also
performed additional controls with no fixative at all, since some epitopes
are particularly sensitive to chemical fixation. We didn´t improve anything
by doing that.
Permeabilization took place for the 15 min of the blocking step,
plus 24h of incubation with the primary antibody. All reagent solutions were
made with 0.1% Triton x-100. We discard the lack of signal being due to
insufficient permeabilization, since other nuclear antibodies can be
perfectly detected with this same protocol. Dilution of the primary antibody
was 1:100, which is in accordance to recommended technical specifications.
Dr. Gaskon Ibarretxe
Submitted Feb 01 2010