[Histonet] RNA DNA (im)possibilities

[Monson, Frederick]

Afternoon Hans,

	I have a long-time favorite which works well for me on sections
of Clarke's fixed material.

	There is no real credit for this except that I know from
experience that it works nicely, AND is fast.

------------Thionin metachromasia of Nucleic Acids 

(Note:  thionin preparations usually contain many related dyes such as
Azure A, B and C.  The point:  Thionin, like methylene blue is not a
pure chemical.  In addition, metachromasia is a strange phenomenon that
does not survive dilute alcohol rinses, thus the absence of 95% or lower
ethanol dilutions.

		Clarke's is:  3(100% ethanol) + 1 (Glacial acetic acid)
		Dye is:  0.1% Thionin (C.I. 52000) in 100 ml glass
distilled water


		Method
       		1.	bring sections to water
			2.	immerse section in dye for 4 min
			3.	rinse in two changes of water (as above)
			4.	immerse in 100% (200 proof, U.S.P.,
undenatured!!) ethanol until no more color is removed (2-3 Coplin jars)
			5.	clear and mount as appropriate

		Result:  DNA blue, RNA purple.
------------------------------------------------------------------------
------------
	A good histochemical procedure that I have used with RNase
(DNase-free) and DNase (RNase free) is as follows:
			Flax and Himes, 1952(Ref if you want it?).
		Prep.
			1.	McIlvaine's Buffer at pH 4.0 (Pearse,
1968)
				a.	0.1M citric acid
122.9ml
				b.	0.2M disodium monobasic
phosphate		 77.1ml

			2.	Azure B (C.I. 52010):  0.25g / ml
McIlvaine's Buffer, adjust to pH 4.0

		Method
			1.	bring sections to water
			2.	treat control sections with appropriate
procedures
			3.	rinse in water (always glass distilled
2x)
			4.	immerse in dye solution for 2 hr at 37oC
			5.	rinse in two changes of water
			6.	immerse in 100% tertiary butyl alcohol
(TBA) for 16 hours
			7.	rinse in 2 changes of TBA for 1 hr each
			8.	mount as appropriate (my Damar mounts
are good after 25 years*)

*	I know!  Nobody cares after 2!

		Result:  DNA crystal azure; RNA shades of purple.

	Methyl Green, Pyronin Y (Kurnick, 1952, J. Stain Technol,
27:233))

		Prep
			1.	0.2% aqueous Methyl Green (C.I. 42585)
extracted with chloroform until no more violet color is observed in the
chloroform partition.
			2.	saturated solution of Pyronin Y (C.I.
45005) in acetone

		Method
			1.	bring sections to water
			2.	immerse in MeGreen solution
				a.	tissues fixed in Clarke's - 15
min
				b.	Zenker acetic and Bouin's - 1 hr
			3.	rinse in two changes of n-butanol
			4.	immerse in pyronin Y for 10-15 sec
			5.	rinse rapidly in acetone
			6.	clear in cedar oil
			7.	clear again and mount as usual (again, I
have always used Damar/Xylene)

		Results:  DNA, green; RNA, pink

Hope this helps,

Fred Monson

Frederick C. Monson, PhD
Center for Advanced Scientific Imaging
Mail to Geology
West Chester University of Pennsylvania
Schmucker II Science Center, Room SS024
South Church Street and Rosedale Avenue
West Chester, PA, 19383
Phone/FAX:  610-738-0437
eMail:  fmonson <@t> wcupa.edu
CASI Page and Scheduling
	http://darwin.wcupa.edu/CASI/


-----Original Message-----
From: Hans Ooms [mailto:j.p.g.ooms <@t> zonnet.nl] 
Sent: Tuesday, January 06, 2004 3:41 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] RNA DNA (im)possibilities


Dear Histonetters,

First of all I Wish you all a prosperous and very healthy New Year!!!!!

Secondly, the following question keeps me busy:

what RELIABLE technique can be used to show RNA and DNA in
paraffinsections ?

Thanks in advance !

Hans Ooms
HT (The Netherlands)
 

 





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