[Histonet] ammonium hydroxide

[Rene J Buesa]

There are somethings I would change:
1- you have to fix all the tissues first
2- those that do require decalcification, treat them separately. Decalcify them not for a specific time period, but until they are decalcified.
3- those tissues not needing decalcification process them just after being fixed. Keeping them in EthOL70 will lead to the problem you have of being too brittle.
4- mouse tissue is very lean and dehydration should be shorter. Ideally with 2-propanol which will dehydrate more slowly and with less shrinkage.
5- I do not like clear-rite. It would be better to just use 2-propanol and substitute the first paraffin station with a mixture at equal parts of 2-propanol + paraffin (2 hours)
6- the 2 following stations with paraffin wax for 2 hours each.
7- the dehydration times you have will be good for 2-propanol but too much for EthOL
8- when the decalcified tissues are ready, process them in the same way as those not requiring decalcification.
René J

--- On Tue, 3/6/12, Canal, Erica <ECanal <@t> hei.org> wrote:


From: Canal, Erica <ECanal <@t> hei.org>
Subject: [Histonet] ammonium hydroxide
To: histonet <@t> lists.utsouthwestern.edu
Date: Tuesday, March 6, 2012, 4:58 PM


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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