[Histonet] PALE MYELIN STAIN

[Mickie Johnson]

Tim,

It might be prudent to try the stain again on a block processed at an
earlier time when the stain was working and see if the results have remained
the same. If it is pale now but wasn't then, then it might be the dye powder
at issue. Has your water changed? Good Luck.

Best Regards,
 
Mickie
 
Mickie Johnson, B.S., HTL(ASCP)
Mohs Histology Consulting Services, LLC
  & Mohs Lab Staffing
2507 S. Manito Blvd.
Spokane, WA 99203
509-954-7134
FAX   509-624-3926
Web: www.mohshistogyconsulting.com & www.mohslabstaffing.com 
Email: mickie25 <@t> netzero.net
 
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-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Tim Wheelock
Sent: Thursday, July 03, 2008 11:07 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] PALE MYELIN STAIN

Hi Everyone:

Lately, I have been having problems with my Luxol Fast Blue myelin stain 
coming out too pale.
I have gone through each step and can't figure out where the problem 
originates.
I even bought another bottle of the dye itself to no avail.

I rechecked and re-made the The Luxol Fast Blue working solution (0.1 
gram Luxol Fast Blue and 1 ml of 10% glacial acetic acid in 100 mls of 
95% ethanol).
I did the same for the differentiator(1% Hydroquinone/5%Sodium Sulfite).
That made no difference at all.
The sections look pale after I remove the excess stain in 95% before the 
differentiator, so I assume it is not a mistake making up the later or a 
problem with the H+ E that I add onto the myelin stain.

I adjusted by increasing the incubation time in a 60C oven from 2 to 4 
hours.
And decreased the time in the differentiator from 2 dips to 1.
This helped a lot, but I knew that these changes did not get to the root 
of the problem.
Yesterday, they still came out pale even with these changes in place

I keep thinking the the problem is in the working solution but am not 
sure how.

(Fixation, processing and sectioning are the same as they have always been.)

I have done this stain for years and never had a problem...until now.

Thanks for any suggestions you may have.



Tim Wheelock
Harvard Brain Tissue Resource Center
203 Mailman Research Center
McLean Hospital
Belmont MA 02478
Phone: 617-855-3592
Fax: 617-855-3199

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