Blue line woes RE: [Histonet] Leica DRS

[Gayle Callis]

Betsy,

Question, not sure why?  but is this occuring with freshly changed 
hematoxylin also?  My RA rep said to pour into staining dish without 
filtering, but I have tried this - and I see all kinds of gunk on my 
slides, so I went back to filtering into dish, and feel more secure the 
blues are gone.

We have used running tap water after hematoxylin for over 26 years, but we 
also live in a part of the USA (Montana) where our water is relatively 
pure, little chlorine, and we never have problems with quality of 
hematoxylin staining no matter what type of hematoxylin we use.  I would 
assume if you use distilled water for rinsing, you would have to change it 
for each run of slides, since it is a static, non-flowing rinse unless 
suggestors (people doing this) have a flowing distilled water setup.

Our rehydration is similar to yours, although we go to 70% alcohol then 
distilled before RA Hematoxylin.
We use RA hematoxylin 1 - 1.5 min
Tap water rinse 1 min and the water flows from the bottom of slides up over 
the top, and the hematoxylin is always cleared away nicely before the next 
step.
Clarifier 1  - 1 min - this is changed daily with big runs
Tap water rinse 1 min
bluing 1 min  this is changed daily with big runs
tap water 1 min
70% alcohol 1 min in place of 95% alcohcol
then proceed to eosin

No background on slides, NOR blue lines, we stain manually and it doesn't 
make any difference what hematoxylin we use

Gayle Callis HTL, HT, MT(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717




At 10:23 AM 8/14/2006, you wrote:
>No tap after the hematoxylin.
>   René J.
>
>"Molinari, Betsy" <BMolinari <@t> heart.thi.tmc.edu> wrote:
>   Get rid of all tap or just the tap rinses after hematoxylin?
>
>Betsy Molinari HT (ASCP)
>Texas Heart Institute
>Cardiovascular Pathology
>6770 Bertner Ave.
>Houston,TX 77030
>832-355-6524
>832-355-6812 (fax)
>
>
>-----Original Message-----
>From: Terri Gillow [mailto:TerriGillow <@t> warrenhospital.org]
>Sent: Monday, August 14, 2006 11:03 AM
>To: Molinari, Betsy
>Subject: RE: [Histonet] Leica DRS
>
>Betsy,
>I feel your pain. The only way we finally got rid of the blue line
>(that caused a lot of blues) is we started using Distilled H2O in place
>of tap water. It has never been back.
>
>-----Original Message-----
>From: histonet-bounces <@t> lists.utsouthwestern.edu
>[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of
>Molinari, Betsy
>Sent: Monday, August 14, 2006 11:54 AM
>To: histonet <@t> lists.utsouthwestern.edu
>Subject: [Histonet] Leica DRS
>
>Hi,
>
>I am frustrated. I have been struggling to solve this problem and now
>turn to you.
>
>My H&E's come out of the stainer with this blue line across them. I
>have juggled times, rinses, solutions.
>
>The slides are cut at 4-5 microns. This is my current protocol
>
>Dryer: 20 min
>
>Xylene x3 5 min
>
>100 % x3 1 min
>
>95% 1 min
>
>rinse in tap
>
>rinse in DH2O
>
>Richard Allan hematoxylin 1 1 min ( initially started w/ hematoxylin 2
>but staining was too dark, I have juggled these times from 15 secs-1
>min)
>
>Rinse in tap 2 min
>
>Richard Allan Clarifier 2 30 secs (have played w/ this time too)
>
>Rinse in tap 2 min
>
>Richard Allan Bluing 1 min
>
>Rinse in tap 2 min
>
>95 % 1 min
>
>Richard Allan Eosin Y 45 secs
>
>100%- Xylenes I min.
>
>The staining is acceptable..good nuclear staining but still that damn
>blue streak! Any suggestions?
>
>Thanks.
>
>
>
>Betsy Molinari HT (ASCP)
>
>Texas Heart Institute
>
>Cardiovascular Pathology
>
>6770 Bertner Ave.
>
>Houston,TX 77030
>
>832-355-6524
>
>832-355-6812 (fax)
>
>
>
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