Re: Histo in Plastic

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From:"Karen D. Larison" <>
Date:Wed, 09 Jun 1999 17:05:18 -0800

We got plenty of those "old" way recipes round here:

Oversight stains for plastic sections

Lee's methylene blue basic fuchsin
	Stock solutions
		1.  0.13 gms methylene blue in 100 ml DW
		2.  0.13 gms basic fuchsin in 100 ml DW
		3.  0.2 M phosphate buffer, pH 7.2 to 8.0

	Staining solution:  Mix
		methylene blue			12 ml
		basic fuchsin			12 ml
		0.2 M PO4 buffer			21 ml
		Ethanol (95% or absolute	)	15 ml
		Filter.  Useful for 4 to 5 days.

	Stain sections 10 to 15 sec.  Remove slides and wash in DW.  Air dry.

	Notes:  Works well with methacrylate sections.  Epon sections may take 	longer or 
may require heating the staining solution.

Humphrey and Pittman   Stain Technology  42:9-14  (1974)
		A.  Methylene blue-azure II  	
			Methylene blue				0.130 gms
			Azure II  (Azure A can be used)		0.020 gms
			Glycerol					10  ml
			Methanol					10 ml
			PO#031#4 buffer, 0.15 M,  pH 6.9		30 ml
			DW						50 ml

		B.  Basic Fuchsin
			Stock solution
				Basic fuchsin			0.100 gm
				50% EtOH				10 ml
			Staining solution
				Stock solution			3 ml
				DW					57 ml

	Staining procedure
		1.  Immerse slides in solution A at 65o C.  Time required varies 			with 
thickness and type of embedding media.  Ten minutes 			works for 7 to 10 um 
sections of Epon or Epon/Araldite. 
		2. Rinse in two changes of DW.
		3. Stain in solution B at room temperature for 20 min.  Again time 			may vary 
with thickness and embedding media. 

		4. Rinse thoroughly in DW.
		5. Dry stained sections on a 40o C. hot plate.   
		6. Sections can be mounted in Permount.  If wrinkling becomes a 			problem try 
mounting in the appropriate resin.  Use just 			enough resin to cover sections.  A 
thick layer of resin may 			prevent focusing at high power if your objective has a 
short 			working distance. 

		1.  Sorensen's phosphate buffer is used in the original method. 
			0.15 M  Na2HPO4		100 ml
			0.15 M KH2PO4		 90 ml
			pH = 6.9

		2.  Authors suggest that stain is stable for up to 4 months.   The 			shelf life 
in our experience is considerably longer. 

		3.  It is difficult to overstain sections.

Date:          Wed, 09 Jun 1999 12:57:18 -0700 (PDT)
From:          Katie B <>
Subject:       Histo in Plastic
To:            Histonet Server <>

Looking for a recipe for Methylene Blue-Basic Fuchsin stain to do in
GMA embedded tissues.  My boss tells me it is the "old" way to do
something like an H+E in plastic.


Catherine "Katie" Bresee Bennett
Laboratory for Experimental Pathology
Department of Veterinary Pathology
Michigan State University

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