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DAPI Staining Protocol Guide

This document provides a protocol for DAPI staining of paraffin embedded tissue sections. It describes making stock solutions of DAPI and McIlvaine's buffer at pH 7.0, as well as a working DAPI staining solution. The protocol outlines steps for dewaxing, rehydrating and equilibrating tissue sections, then applying the DAPI solution for 15 minutes before mounting and observing under UV light. It also describes a method for counterstaining using Gel Mount containing DAPI.

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355 views1 page

DAPI Staining Protocol Guide

This document provides a protocol for DAPI staining of paraffin embedded tissue sections. It describes making stock solutions of DAPI and McIlvaine's buffer at pH 7.0, as well as a working DAPI staining solution. The protocol outlines steps for dewaxing, rehydrating and equilibrating tissue sections, then applying the DAPI solution for 15 minutes before mounting and observing under UV light. It also describes a method for counterstaining using Gel Mount containing DAPI.

Uploaded by

surendrasrawan
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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DAPI STAINING PROTOCOL
DAPI 4,6 diamino-2-phenylindole2HCl

DAPI staining is specific at pH 7, at other pH non-specific staining occurs.

1000X DAPI stock
- dissolve 0.2 mg DAPI in 1 ml dist. H
2
O.
- store at 4C in dark
McIlvaines buffer pH 7.0
- stock solution A: 0.1 M citric acid for 200 ml use 4.2 g
- stock solution B: 0.2 M Na
2
HPO
4
for 800 ml use 22.7 g

Mix 2 parts of stock A and 8 parts of stock B to make pH 7.0 buffer.
DAPI staining solution (600 nM DAPI)
- 10 ml McIlvaines pH 7.0
- 10 l 1000X DAPI stock

Staining of paraffin embedded sections:
Use Coplin jars for the following steps until DAPI staining. Dewax sections in xylene (2 x 5
min), rehydrate in ethanol series (absolute, 95% for 5 min, 70%, 30% ethanol, dH
2
O for 3 min),
equilibrate in McIlvaines buffer (5 min, prepared fresh). Drain slides, put them on paper towel,
apply DAPI staining solution on slides (~200 l), incubate 15 min in dark (cover with a box),
drain excess solution, mount coverslips with Gel Mount (or other aqueous mounting medium).
Observe with UV excitation, nuclei will emit cold blue fluorescence.

Counterstaining:
Gel Mount DAPI
- 1 ml Gel Mount
- 1 l 1000X DAPI stock
Mix thoroughly, but take care not to create too much bubbles.

Use DAPI staining as a final step in any fluorescent staining procedure. If the final wash is in a
buffer with a pH close to 7, such as PBS, equilibration is not necessary, otherwise equilibrate in
McIlvaines buffer for 5 min. Drain excess solution from the slide. Apply two separate drops of
Gel Mount DAPI on the sections, then carefully lower the coverslip on sections. Let stand in
dark for few minutes, then observe DAPI stain with UV excitation.

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