[Histonet] RE: ammonium hydroxide

[Andrea Ferullo (non-Celgene)]

You can try removing two of the absolute alcohol steps, as it seems you are using too much 100% etoh.  Also, the tissue is probably dry from not being infiltrated properly, so instead of doing 3 paraffin steps, you can try 1 step for 1 hour to remove xylene, and then the second step over night.  This should penetrate deep in the tissue and should help with the dryness.

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Rathborne, Toni
Sent: Friday, March 09, 2012 2:42 PM
To: 'Canal, Erica'; histonet <@t> lists.utsouthwestern.edu
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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