[Histonet] RE: ammonium hydroxide

[mohamed abd el razik]

From  my experience it is better to fix all the samples in NBF to coagulate the prteins and keep the normal morphology... then we could transfer the samples to 70% ethanol and no matter how long it will stay as it is considered a fixtave. 
In your case the proplem is that the processing time is too long in absolute eth. changes as it cause dryness of tissue and become difficulte to cut. The over all time in absolute eth. in our lab not exceed 80 mins. for mouse small samples. Also i recommed that you begain with 70% then 80% then 95% followed by absolute changes and the rest of your program
 
hope that help
 
Mohamed Abd El razik
Ass. Lec. of Cytology & Histology
Faculty of Vet. Med.
cairo University- Egypt


________________________________
From: "Rathborne, Toni" <trathborne <@t> somerset-healthcare.com>
To: "'Canal, Erica'" <ECanal <@t> hei.org>; "histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu> 
Sent: Friday, March 9, 2012 9:42 PM
Subject: [Histonet] RE: ammonium hydroxide

Although it has been many years since I worked in research, I never remember placing tissue in etoh overnight or longer. 
I would suggest that if there are any mice that are extras and don't make it into a study, that you experiment with different protocols using these mice. I'm sure you'll get more suggestions from those currently in the field. Good luck!

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Canal, Erica
Sent: Tuesday, March 06, 2012 4:59 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] ammonium hydroxide

Hello, I am a newly certified histotech, so I have lots to learn but I would like to know what you experts think about our tissue processing.

We process mouse tissue.  Tissue is fixed in nbf, and then the skull and spine is placed in decal soln. overnight while the other tissue organs etc,; are placed in 70% etoh over night for a day even two sometimes.
Then when it comes to processing here is our pgm. :

70% etoh ...........15 mins

95% etoh ...........45 mins

Absolute etoh....60 mins

Absolute etoh ...60mins

Absolute etoh....60mins

Absolute etoh....60 mins

Absolute etoh...60 mins

Clear rite..........2hrs

Clear rite .......2 hrs 15mins

Clear rite.......2hrs 30 mins

Histowax........1hr

Histowax.........1hr

Histowax........1hr 30 mins



So after processing my tissues are very very dry and brittle. I soak them on ice with soapy water and sometimes use ammonium hydroxide in the ice water, to soften my tissue. I would like to know if you have any suggestions for my processing cycle and I would like to know If ammonium hydroxide affects IHC staining.

Thanks much...histo newbie 



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