[Histonet] bluing

[Lee & Peggy Wenk]

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 <@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!!



  ________________________________
This e-mail and any files transmitted with it are confidential and are 
intended solely for the use of the individual or entity to whom they are 
addressed. If you are not the intended recipient or the individual 
responsible for delivering the e-mail to the intended recipient, please be 
advised that you have received this e-mail in error and that any use, 
dissemination, forwarding, printing, or copying of this e-mail is strictly 
prohibited.

If you have received this e-mail in error, please immediately notify the 
HealthPartners Support Center by telephone at (952) 967-6600. You will be 
reimbursed for reasonable costs incurred in notifying us. HealthPartners 
R001.0
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet