Immunofluorescence Counterstain - DAPI Counterstain Solution

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Tony Rook
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Immunofluorescence Counterstain - DAPI Counterstain Solution

Please find the following media preparation method:

Immunofluorescence Counterstain:

DAPI Counterstain Solution

DAPI is a popular nuclear counterstain for use in multicolor fluorescent techniques. Its blue fluorescence stands out in vivid contrast to green, yellow or red fluorescent probes of other structures. DAPI stains nuclei specifically, with little or no cytoplasmic labeling. The following protocol is used for staining of tissue sections or for staining cultured cells on slides.

DAPI Stock Solution (5mg/ml or 14.3 mM):

DAPI (Molecular Probes, Cat# D-1306) -------------- 10 mg

Dimethylformamide (DMF) ------------------------------ 2 ml

Mix to dissolve and it may take sometime to completely dissolve.

Aliquot and store in 20 C.

DAPI Working Solution (100ng/ml or 300 nM in PBS):

DAPI stock solution ---------------------------------------------- 2 ul

PBS ------------------------------------------------------------ 100 ml

Store this solution at 4 C in brown bottle or wrapped with aluminum foil to protect from light (to achieve stronger staining, use 4 ul stock solution or reduce PBS amount to 50 ml).

Incubate sections in dark for 30 minutes at room temperature.

Staining Pattern: Nuclei will be stained bright blue.

Suggested Use

Counterstain for immunofluorescence when green (FITC) or red (Texas Red) fluorescent marker is used.

Link Reference:
http://www.ihcworld.com/_protocols/counterstain_solutions/DAPI.htm

Fraser Moss
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Dapi willl stain cultured

Dapi willl stain cultured cells in 30 seconds. Sections may take a little while longer, but you don't need to wait 30 minutes

pkhanna
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Hi all

Hi all
I have to stain paraffine embedded sections with DAPI and counterstain with FITC. I don't know how to stain with FITC. kindly let me know the detailed protocol.

samm
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pkhanna wrote:Hi all

pkhanna wrote:

Hi all
I have to stain paraffine embedded sections with DAPI and counterstain with FITC. I don't know how to stain with FITC. kindly let me know the detailed protocol.

Check out the protocol for Ab staining on paraffin sections present on the site.
Basically you have to
1) Hydrate
2) Perform Ag retrieval (Ag/Ab dependent)
3) Neutralize endogenous fluorescence
4) Permeablize and block
5) Stain with primary Ab
6) If necessary, stain with appropriate secondary AB/tertiary rgts (e.g.Streptavidin)
7) DAPI stain
8) (Post-fix/Dehydrate - opt) Mount in anti-fade containing mounting medium.