2/23/98

Recently returning to the EM fold after an absence of several years, I

have been unhappy with the staining of LM sections with Toluidine

Blue/Borax or Tolouidine Blue/Basic Fuschin.Buried in a cupboard however

I found a bottle labelled Richardsons Stain which seems to work very

well. However I have no idea whats in it, (its either Toluidine Blue or

Methylene Blue based).I am loathe and embarressed to admit that the

bottle is labelled in my own handwriting, however advancing years and

alzheimers have combined to defeat my memory. Any help would be much

appreciated.

Peter Smith

AgResearch Wallaceville

Upper Hutt

New Zealand

smithp@agresearch.cri.nz

Richardson's stain is 1% Methylene Blue made up in 1% borax, mixed in equal

parts with 1% Azure II. You have to filter it to use it. Hope this helps.



Lesley Weston.

lesley@unixg.ubc.ca

Dear Peter,

I found the Richardsons Stain in P. Boecks book "Der Semidünnschnitt":



1% Azur II in A.dest

1% Methyleneblue in 1% Borax

mixed 1+1



Dry sections, staine for 1-2 minutes at 60 degrees (hetating plate), rinse

slides in A. dest, drying and mounting (immersion oil or whatever).



The original method is described in:

Richardson KC, Jarett L, Finke EH (1960) Embedding in epoxy resins for

ultrathin sectioning in electron microscopy. Stain Technol. 35: 313-325



Probably there are some modifications of this method, but I hope this helps.



Jens





---------------------------------------------------------------

Dr. Jens Buecking Tel. +49-(0)421-218 3745

University of Bremen Fax. +49-(0)421-218 4620

Dep. of Biology Email jbueck@biologie.uni-bremen.de

Leobener Str. - NW2

28359 Bremen

Hey Folks --



Richardson's Stain:



Stock 1: 1% Azure II in ddH2O.



Stock 2: 1% Methylene Blue in 1% Borax (Sodium Borate)



Mix stocks 1:1 and store in a syringe with 0.22um filter.



Protocol for 0.5 um EPON or LRGold sections:



1) Transfer sections to drop of ddH2O on a glass slide and warm on

a hot plate until

water has evaporated.



2) Cover the still-warm sections with stain for 10-30 seconds.



3) Rinse with a stream of ddH2O.



4) Dry sections with a jet of air, then use a KimWhipe to dry the

entire slide.



5) Mount in immersion oil.



I've also used this stain on 0.1um cryosections used for orientation prior

to cutting cryoultrathins.

Greg Martin

Light/Confocal Microscopy and Image Analysis Specialist

Biological Imaging Service

The Jackson Laboratory

TJL Box 43

600 Main Street

Bar Harbor. ME 04609



207-288-6191



gvm@aretha.jax.org

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