[Histonet] RE: Bluing

[gayle callis]

Dear All, 
 
We use commercial bluing reagents for convenience e.g. Richard Allan Bluing
Reagent ready to use and have used Scotts Tap Water Substitute Concentrate
diluted before use.  We do not use ammonia water anymore as the high pH can
cause section lift off from slide.  Here is recipe for Scotts. 
 

SCOTT'S TAP WATER SUBSTITUTE (You can make this up as a 10X concentrate if
you wish.  Store in house concentrate at 4C or growth will occur) 

Sodium bicarbonate                            2  g

Magnesium sulfate (anhydrous)      20  g

Tap Water                                           1 L

 
Peggy presented a good explanation of bluing.  We do not use tap water
anymore due to incorrect pH.   One of the failures for complete bluing is
not changing the bluing reagent daily with large volumes of slides. Lower
number of slides, one might be able to get by with fewer changes of bluing.
Tap water rinses dilutes the reagent and can change the pH, which should be
pH 8. If there is a large volume of slides, anything over 75 or more, we
change the bluing reagent daily. It is important to remember that Clarifiers
e.g. acetic acid reagents used with progressive hematoxylins (Richard Allan
Hematoxylin 1, Gill 1,2,3) will suffer from tap water rinse dilution too. We
also change those, and if Clarifier looks rather yellow, then you have
probably exhausted the acetic acid and its effectiveness.  Jerry Fredenburgh
has given excellent workshops on hematoxylin staining in the past and passed
on many of these facts. Consequently, we have no clarifying or bluing
problems.  
 
Fredenburgh recommendations for Richard Allan Hematoxylin 1 staining
procedure that we use: 
Hematoxylin 1 - 1.5 min, longer if needed
Tap water rinse-1 minute
Clarifier - 1 minute
Tap water rinse - 1 minute
Bluing - 1 minute
Tap water rinse - 1 minute
 
Remember, static or non-running water rinse stations are contaminated with
carryover hematoxylin, clarifier and/or bluing reagent.  If you do NOT
remove the bluing reagent with a good rinse, then eosin staining is affected
by cations carried over from the bluing reagent.  We also have an alcohol
step (matching % of alcohol in eosin) before going into this stain.  This
helps remove the cations plus prepares the section for eosin staining.
Perform good rinses and change static water rinse stations after a run, or
frequently during a day of heavy staining.  The second and third racks of
slides on a stainer should have the same tap water rinse conditions as the
first rack.   Yes, extra work but you need to ensure consistent staining
results of H&E of ALL sections- the universal workhorse stain we depend on
so heavily.     
 
Years ago, Richard Allan had an excellent manual for H&E staining guidelines
on how to perform and adjust your H&E staining.  Sadly the manual is no
longer published but the good news is ......  I saved my copy, and scanned
it to pdf if anyone wants it.  
 
Gayle M. Callis
HTL/HT/MT(ASCP)
Bozeman MT 
 
 
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Peggy Wenk wrote: 
 
It might just need longer time in the bluing agent.
 
Hematoxylin solutions stain the nuclei reddish (look at your slide right 
after coming out of hematoxylin - tissue will look reddish). The alkaline 
solutions that are used for "bluing" remove a H+ group on the aluminum 
hematein (that's the staining chemical of the hematoxylin solution), and 
change it to a -OH group. This changes the aluminum hematein from a reddish 
color to a blue color.
 
The more alkaline (higher pH) the bluing agent, the faster this reaction and

color change, and the less time is needed in the bluing agent. The lower the

alkalinity (not as high pH but still in the alkaline range) the bluing 
agent, the slower the reaction and color change, and more time is needed in 
the bluing agent. Dilute ammonia water usually only takes a few seconds, as 
it's pH is usually high (pH 10). Tap water can have a pH of around 7, and 
may take 5-10 minutes to "blue". If the tap water is more acidic (pH 5 or 
below), the slides may not "blue".
 
Now, we have to get all the nuclei from the reddish color to the bluish 
color. If the slide is not in the bluing agent for enough time (for the 
type/pH of bluing agent), then some of the nuclei change to blue, while some

still remain reddish (or within a single nucleus, some of the DNA has 
changed to blue, some remains reddish), hence a more purple color.
 
So, the easiest thing to try right now is to stain 2 slides from the same 
block (serial sections) in your hematoxylin for the same time, put one in 
the bluing agent for the usual time, put the other in the alkaline solution 
for extended time, and see if the nuclei on the second slide are now more 
blue than the first slide.
 
Let us know.
 
Peggy A. Wenk, HTL(ASCP)SLS
Beaumont Hospital
Royal Oak, MI 48073
 
-----Original Message----- 
From: Webb, Dorothy L
Sent: Friday, June 24, 2011 3:20 PM
To: 'histonet  <http://lists.utsouthwestern.edu/mailman/listinfo/histonet>
<@t> lists.utsouthwestern.edu'
Subject: [Histonet] bluing
 
Looking to change my "bluing" step in the H&E process to obtain a bluer 
(less purple) hue to the nuclear detail.  What is everyone using in their 
bluing step??
 
 
Thanks for all of your ideas!!