RE: Hoechst in tissue sections

From:Patsy.Ruegg@UCHSC.edu

Reference: Cesar La Barca and Kenneth Paigen
A Simple~ Rapid~ and Sensitive DNA Assay Procedure. Analytical Biochern 102,
344-352.
I. Stock Solutions
	A.	Hoechst Dye Stock Concentrate
0.5 mg/mi Hoechst dye in water.
Store protected from light, at 4C, for up to 6 months. Working Solution: 4
ul in 10 mi 4.6 M NaCI-PO4 buffer. Make up fresh before each use.
B.	DNA STANDARD STOCK
	5 mg calf thymus DNA in 10 mi 5 mM NaOH
	Allow to stir overnight at 4C, pipet to further dissolve. Aliquot
into 10 mi sterile tubes (200 ul) and store at -20C.
C.	4.6 M NaCI-PO4 buffer
	1.14 g/L NaH2PO4 (anhydrous)
	5.753 g/L Na2HPO4 (anhydrous) 268.6 g/L NaCI
	Adjust pH to 7.4
	D. 1M NaOH
	E. 1M HCI+200mM PO4
	500 mi X (50 mM NaH2PO4)
	(150 mM Na2HPO4) Adjust pH to 7.4
	Discard 41.67 mi of buffer, adjust volume to 500 mls with 12 M HCI.
	VERY IMPORTANT
	It is now necessary to ensure that the NaOH and
1M HCI+200mM PO4 are of equal molarities. To a 50 ml tube, add 5 mis of
each, and check the pH. If other than pH 7.4 adjust concentrations until
result of the addition is 7.4.
F. 0.25 M NaCI
." ".;."'..""c."""'",..,,
II. CELL PREPARATION
A. Aliquot 100 ul sample ~to 1.5 mI tubes. Add 200 ul 1 M NaOH. Vortex well
and freeze at -20C. Incubate 1 hr at room temperature, or overnight at 4C.
B. Add 200 ullM HCI+200mM PO4. Sample is now ready.  III. PREPARATION OF DNA
STANDARD CURVE A. DNA SOLUTION A Remove 1 tube of DNA stock solution from
-20C freezer. Add 1.9 mIIM HCI+200mM PO4 and 1.9 mI NaOH. B. DNA SOLUTION B
Tol.6 mI 0.25 M NaCI, add 400 ul DNA SOLUTION A.
IV .DNA ST ANDARD CURVE
Add directly to microflour 96 well plate the following amounts:
DNA final concentration 0.25 M NaCI DNA A ~NA B
3.2 ug/mI 93 ul 32 ul
1.6 ug/mI 109 ul 16 ul 0.8 ug/mI 117 ul 8 ul
0.4 ug/mI 105 ul 20 ul 0.2 ug/mI 115 ul 10 ul 0.1 ug/ml 120 ul 5 ul 0.0
ug/mI 125 ul You must load plate in dupicate only, starting from highest
concentration and going to lowest, in 125 ul volumes.  A. Add 125 ul of
sample into remaining wells, in duplicate.
B. Add 125 ul Hoechst dye (of working concentration) to each well.  Incubate
in light protected environment, for at least 20, but less than 60 minutes C.
Read on Dynatech Microflour plate reader for relative flourescence units.
Estimate the DNA content of samples from standard curve.

Patsy Ruegg



		-----Original Message-----
		From:	Joshua Griffiths
[mailto:joshua.griffiths@adelaide.edu.au]
		Sent:	Tuesday, July 31, 2001 4:57 PM
		To:	histonet@pathology.swmed.edu
		Subject:	Hoechst in tissue sections

		Hi everyone,
		I am very much a novice in histology. Can anyone help me to
stain for
		apoptosis in paraffin embedded lung tissue sections to
quantify cell
		death levels? I have tried TUNEL and was less than impressed
with the
		results. I would like to try Hoechst with propidium iodide
as a counter
		stain. Does anyone have a protocol for this as most of the
litera

<< Previous Message | Next Message >>