RE: [Histonet] Re: Distilled water rinse versus tap water rinsing, a curious thing

From:"Margaryan, Naira"

Dear Gayle/Chris/Andrea,


 I usually use hematoxylin Mayer's as a counterstain and after wash with
H2O, 95%Alcohol, 2x100% Alcohol and Xyline for immuno-.


The "Scott's Tap Water" and bluing solution I use for H&E only.


If it is wrong, please, let me know. Why you use "Scott's Tap Water' in






-----Original Message-----
[] On Behalf Of Andrea
T. Hooper
Sent: Tuesday, April 26, 2005 4:54 PM
To: Gayle Callis;
Subject: [Histonet] Re: Distilled water rinse versus tap water rinsing,a
curious thing


Dear Gayle/Chris --


Very interesting indeed ... have you published these findings? It 

would be interesting to know the regional differences in such 

typically benign steps as washing after the protocol is complete! It 

is interesting to ponder that such steps could be responsible for 

such variations from lab to lab.


I have found that for "delicate" sections - such as mouse 

hematopoietic tissue cryosections which have been fixed with acetone 

- that the crucial "make it or break it" step is what you blue in 

after hematoxylin counterstain. Classic ammonia water (0.25% ammonium 

hydroxide in dH20) seems to be incredibly damaging to sections so 

instead I use "Scott's Tap Water Substitute" from Sigma. I think 

someone else (maybe one of you guys) mentioned this recently as well 

on Histonet.


Have a great week,



At 12:48 PM -0600 4/26/05, Gayle Callis wrote:

>Andrea and Chris,


>I have seen Chris's results on rinsing.    We collaborated on this 

>after I got home from visiting his lab.  I repeated his rinsing 

>experiments in my lab to see if I had the same problems. I did NOT 

>get the same results he observed.   Could this be caused by 

>differences in another countries water and purification systems? 

>Very curious why this happens as rinsing with distilled water is a 

>very common practice.


>My experience with acetone or acetone/ethyl alcohol combo fixed 

>mouse frozen section IHC is the same as Andrea's, no problems or 

>damage to sections after either rinsing with MilliQ, RO or tap 

>water.  Acetone fixed sections are less robust than the AA combo 

>fixed sections, but distilled water rinse was not a problem anyway. 

>We do not use NBF for murine IHC work here.





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