[Histonet] Histonet Digest, Vol 140, Issue 5

Tue Jul 7 13:06:29 CDT 2015

Alkaline Phosphatse bubbles

Hi Tim,
Use Dako ultramount. The bubbles are caused by the naphthyl breaking down
and releasing CO2 under the coverslip. Apply small amount of the ultramount
to cover the muscle tissue and bake in 65 degree oven of 15 to 20 minutes.
If you bake it too long the myofibers will draw up and damage the
morphology of the section.
Kevin
On Jul 7, 2015 1:29 PM, <histonet-request at lists.utsouthwestern.edu> wrote:

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> Today's Topics:
>
>    1. Specimens lost during processing. (STEVEN PINHEIRO)
>    2. Preventing Bubbles in Alkaline Phosphatase (Morken, Timothy)
>    3. Histology on Rat spine. (Clough, Bret)
>    4. 12ml tubes for Dako IHC stainers (Sally Price)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Mon, 6 Jul 2015 18:28:21 +0000
> From: STEVEN PINHEIRO <SPINHEIRO at lumc.edu>
> To: "histonet at lists.utsouthwestern.edu"
>         <histonet at lists.utsouthwestern.edu>
> Subject: [Histonet] Specimens lost during processing.
> Message-ID:
>         <
> B06172B2B6AB254DB7DA4F8DBF110042801D69F3 at SB01MSTMBX01.sb.trinity-health.org
> >
>
> Content-Type: text/plain; charset="us-ascii"
>
> All,
> Looking for help in analyzing the entire scope of the process. There is
> not much published data (that I can find) and I am hoping this group can
> lend some expertise.
> Our rate is higher than we would like it to be. There is no consistent
> size at risk although GI and Derm biopsies are the biggest involved group.
> We have broken it down into steps.
>
> 1.       Can be lost at grossing- either never loaded into the cassette at
> all, or cassette was discarded. Thus we hold on to our waste and can search
> for misdirected cassettes if need be.
>
> 2.       Lost in the processor itself. Most are wrapped. If large enough
> not to be wrapped, we would not expect the processor to eat them, so assume
> cassette lid not properly closed. Frankly the highest number of losses
> we're seeing is no tissue found in cassette by embedders.
>
> 3.       I am being told that we can't use micromesh cassettes in our
> microwave processors (Milestone Pathos) and want to know if anyone is.
>
> 4.       Tissue not seen at embedding. Again no way to tell when the
> tissue disappeared. We know that tiny tissue can spring out during the
> opening at embedding but I don't know how else to examine or limit this
> step.
>
> 5.       Tissue can be exhausted during microtomy. Rare but noteworthy.
> I am hoping people can tell me about their procedures for dealing with
> "specimens that don't survive processing", what safeguards they have in
> place, and to some extent what your own lab percentage  or experience is.
> Apologies in advance for the length of the message, but could really use
> your help.
>
>
> Steven Pinheiro, MBA, MLS(ASCP)DLMCM
> Manager Anatomic Pathology and Cytology
> Loyola University Medical Center
> 2160 S First Ave, Bldg 110 Rm 2214
> Maywood, IL 60153
> 708-327-2642 (O)
> 708-327-2620 (F)
> spinheiro at lumc.edu<mailto:spinheiro at lumc.edu>
>
> "You must do the thing you think you cannot do"
>    E. Roosevelt
>
>
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>
> ------------------------------
>
> Message: 2
> Date: Mon, 6 Jul 2015 19:39:35 +0000
> From: "Morken, Timothy" <Timothy.Morken at ucsf.edu>
> To: Histonet <histonet at lists.utsouthwestern.edu>
> Subject: [Histonet] Preventing Bubbles in Alkaline Phosphatase
> Message-ID:
>         <761E2B5697F795489C8710BCC72141FF602F63AC at ex07.net.ucsf.edu>
> Content-Type: text/plain; charset="iso-8859-1"
>
> All knowing Histonet....
>
> We started doing alkaline phosphatase on muscle frozen a little while ago
> and have had an issue with apparent air bubbles forming over the tissue.
> Not trapped air from coverslipping, but forming from apparent reaction in
> the tissue. Does anyone have experience with this and a solution?
>
> Images:
> http://histosearch.com/imageupload/alkaline-phosphatase-bubbles/
>
> We tried longer formalin fixation after the stain (10 min) and an acetic
> acid rinse after the tap water, after the formalin. Still the same problem
>
> The only mention I have found about this is a Histochemistry text by Lojda
> from 1979 that says to fix in formalin for several hours after staining to
> prevent bubble formation. Does anyone have anything shorter? I don't really
> remember seeing this when I did these stains 20 years ago....
>
>
>
> Our procedure (from a method given to our neuropath folks by a group in
> Australia):
>
> Frozen sections of muscle
>
>
> Borate Buffer, pH 8.8:
>    0.992 g Boric Acid
>    2.28 g Sodium Tetraborate
>    200 ml distilled water
>    Mix well. Adjust to pH 8.8. Store at 4?C.
>
> 0.1M Magnesium Sulphate
>    0.6 g Magnesium sulphate, anhydrous (M7506-500G)
>    50 ml distilled water
>    Store at 4?C.
>
> ALP Incubation Solution:
>    15 ml Borate Buffer
>      2 ml 0.1M Magnesium Sulphate
>    16.5 mg 1-naphthyl phosphate
>    16.5 mg Fast Blue RR
>    Mix in well order. Filter.
>
> Glycerin Jelly Mounting Media
>
>
> 1.     Place glycerin jelly in 60?C oven to liquify
> 2.     Air dry slides 15 minutes.
> 3.     Incubate in filtered ALP Incubation Solution at 37?C...45 minutes.
> 4.     Rinse in tap water.
> 5.     Change to 10% formalin...1 min
> 6.     Rinse in tap water.
> 7.     Air dry.
> 8.     Coverslip with Glycerin Jelly.
>
>
> Tim Morken
> Pathology Site Manager, Parnassus
> Supervisor, Electron Microscopy/Neuromuscular Special Studies
> Department of Pathology
> UC San Francisco Medical Center
> 505 Parnassus Ave, Box 1656
> Room S570
> San Francisco, CA 94143
>
> (415) 353-1266 (ph)
> (415) 514-3403 (fax)
> tim.morken at ucsfmedctr.org<mailto:tim.morken at ucsfmedctr.org>
>
>
>
> ------------------------------
>
> Message: 3
> Date: Tue, 7 Jul 2015 03:05:14 +0000
> From: "Clough, Bret" <Clough at medicine.tamhsc.edu>
> To: "Histonet list serv. (histonet at lists.utsouthwestern.edu)"
>         <histonet at lists.utsouthwestern.edu>
> Subject: [Histonet] Histology on Rat spine.
> Message-ID:
>         <28529330310DFE4A9F6FFF708AEB090C552CC8CA at CSR-Mail1.ad.tamhsc.edu>
> Content-Type: text/plain; charset="iso-8859-1"
>
> Hello everyone,
>
>
>
> I have some rat spines that I need to decal, process, and embed for
> sectioning. Is there someone in the histonet community that would be
> willing to either share with me their protocol or at least give me guidance
> with these spines. These are larger than any tissue that I have ever had to
> process. The spines have been fixed in Carson's fixative. Any help would
> greatly be appreciated.
>
>
>
> Sincerely,
>
>    Bret Clough
>
>    Texas A&M Health Science Center
>
>    Temple, Texas
>
>
> ------------------------------
>
> Message: 4
> Date: Tue, 7 Jul 2015 12:29:43 -0400
> From: Sally Price <sprice2003 at gmail.com>
> To: histonet at lists.utsouthwestern.edu
> Subject: [Histonet] 12ml tubes for Dako IHC stainers
> Message-ID:
>         <
> CAHonC4Bnfypr5XUL3o92B3VtTjACKrbRPBLs5YcGSkOV9GsGUg at mail.gmail.com>
> Content-Type: text/plain; charset=UTF-8
>
> Can anyone recommend a vendor for the 12ml tubes that are used on original
> Dako stainers other than Dako?
>
>
>
> --
> Sally Price
>
>
> ------------------------------
>
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> ------------------------------
>
> End of Histonet Digest, Vol 140, Issue 5
> ****************************************
>