[Histonet] RE: ammonium hydroxide - long response

[Elizabeth Chlipala]

Erica

What kind of decal are you using? we use 10% Formic Acid, and I don't believe that a mouse skull or spine would decal properly over night unless it's a HCL acid based decal solution.  So the issue may be that your samples are not adequately decaled to begin with.  If you are processing soft tissue on this cycle its way too long.  Here is an example of our processing cycle for mouse soft tissue and we would have a separate processing cycle for the mouse bone samples.  In my opinion the samples need to processed separately, we also process mouse skin on the longer processing cycle too.  For rodent soft tissue I find that after the tissue is grossed in it must be placed back into 10% NBF. If you gross and place the tissue in 70% alcohol you are going to run into problems with sectioning and the tissue will crack once the slides are stained.

For soft tissue here is our processing cycle, its basically 20 minutes per station for mouse soft tissue and 30 minutes per station for rat soft tissue. If you are using a xylene substitute you will need 3 changes of that instead of 2 changes of xylene, we are now using propar for most of our rodent soft tissue samples.  For mouse and rat eye samples we do not let the samples sit on the processor overnight we start the processor in the morning we use the 20 minutes cycle for both mouse and rat eyes. In the research setting I find that we will create a specific processing cycle for some tissue types and we have over 12 different cycles that we use routinely.  I know of some labs that have over 30 different processing cycles.

50% alcohol     20 min          50% alcohol     20 min
70% alcohol             20 min          70% alcohol             20 min
80% alcohol             20 min          80% alcohol             20 min
95% alcohol             20 min          95% alcohol             20 min
100% alcohol    20 min          100% alcohol    20 min
100% alcohol    20 min          100% alcohol    20 min
Propar          20 min          Xylene          20 min
Propar          20 min          Xylene          20 min
Propar          20 min          Paraffin                20 min
Paraffin                20 min          Paraffin                20 min
Paraffin                20 min          Paraffin                20 min
Paraffin                20 min

Bone and Skin samples - most species

For bone and skin we have found that longer processing cycles are ideal and we have not really encountered any issues with over processing, some might think that this cycle is too long but it our hands it works great.  We use three absolutes and 3 xylenes and 4 paraffins for bone and skin samples for most species but we will even go longer if required for some tissue samples.

50% alcohol     60 min
70% alcohol             60 min
80% alcohol             60 min
95% alcohol             60 min
100% alcohol    60 min
100% alcohol    60 min
100% alcohol    60 min
Xylene          60 min
Xylene          60 min
Xylene          60 min
Paraffin                60 min
Paraffin                60 min
Paraffin                60 min
Paraffin                60 min

Good Luck

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308-1592
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
www.premierlab.com

Ship to address:

1567 Skyway Drive, Unit E
Longmont, CO 80504

-----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 2:59 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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