[Histonet] Alkaline NaCl saturated Congo Red Procedure (Puchtler et al. 1962)

[Bryan Llewellyn]

Congo red (and similar dyes) stains amyloid by hydrogen bonding.  It can 
also combine with other tissue components ionically since it is an acid 
dye.  Using an alkaline solvent reduces ionic bonding but permits 
hydrogen bonding to go ahead.  The amyloid will stain regardless of 
whether you add alkali or not, but eosinophilic (congoredophilic?) 
components may well stain pink and cause confusion since they will be 
the same colour as the amyloid (but will not display green 
birefringence).  With the alkali you are assured that only amyloid (and 
possibly eosinophils and paneth cell granules) will be pink.

Incidentally, the solvent is ethanolic.  This is to reduce the polarity 
of the solution and thereby inhibit ionic bonding while promoting 
hydrogen bonding.  The ethanolic solvent in conjunction with the alkali 
is what causes the specificity of the technique.  Changing either would 
affect the results to some degree.  If you left out the alkali you would 
likely notice a difference in the long term.

Bryan Llewellyn



Joachim.Velden <@t> evotec-neurosciences.com wrote:
> Dear Histonet Members,
> 
> could someone please explain to me the purpose/effect of alkalinization by 
> adding 1 ml of 250 mM NaOH to 100 ml of the staining solution used in 
> Puchtler's alkaline, NaCl-saturated Congo Red staining method?
> Accidentally, I once forgot to add the NaOH, but the staining results did 
> not differ from those that we usually obtain when we stick to the 
> alkalinizing protocol. Can one omit the alkalinization without doing any 
> harm to the method's nice sensitivity and specificity towards amyloid 
> and/or reproducibility of staining results? What is the alkalinization 
> intended to be good for?
> This question is aimed at some practical consequences: Omitting the 
> alkalinization step would (i) save time, (ii) spare using corrosive agent 
> (NaOH), (iii) leave the staining solution more stable (than for less than 
> one day with NaOH added) and thus (iv) save staining reagents 
> (particularly expensive ethanol) and congo red dye.
> 
> Thank you for your expert opinion.
> 
> Best regards,
> 
> Joachim
> 
> 
> 
> -----------------------------------------------------------------------------------------
> Dr. Joachim Velden
> Evotec Neurosciences GmbH
> Schnackenburgallee 114
> D-22525 Hamburg
> Germany
> 
> Phone +49-40-56081-394        Fax +49-40-56081-222
> joachim.velden <@t> evotec-neurosciences.com
> http://www.evotec-neurosciences.com
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