[Histonet] Tissue Adherence Issue

[Dorothy Hu via Histonet]

>
>  2. Tissue Adherence Issue (Knutson, Deanne)
>

Hello,

I think your testing tissue might be over or under fixed in formaldehyde
fixative?
You didn't tell what kind of tissue though. We deal with bone tissue which
needs extensively bake (at 37, 46 degree over night and 58 one hour) and
carefully handled. Superfrost Plus slides are used. Especially gentle heat
for heat induced antigen retrieval. Try to use enzyme antigen retrieval as
possible.
Best luck.

Dorothy Hu



> Message: 2
> Date: Wed, 17 Jul 2019 09:21:49 -0500
> From: "Knutson, Deanne" 
> To: "'histonet@lists.utsouthwestern.edu'"
> 
> Subject: [Histonet] Tissue Adherence Issue
> Message-ID:
> <
> 1e0e2b14c709174b8ac2be0ae7f768330159b5756...@exchange2k7.staprimecare.org>
>
> Content-Type: text/plain; charset="us-ascii"
>
> Fellow Histonetters -
>
> I would appreciate your feedback on an intermittent issue that has shown
> up in our lab.
> All of a sudden, we are having difficulty with tissue specimens falling
> off of our slides on the IHC stains and special stains sporadically.
> About a year ago, we switched instruments on the IHC bench from the Leica
> BOND to the ROCHE ULTRA, and we use the Ventana NexES special stainer.
> No adherence issues with all of the validation slides that were run.
> We have tried various types of slides recently as well, and the issue
> still prevails.
>
> Would any of you mind telling me your slide workflow - type of slide used,
> gelatin or not used in flotation bath, how long slides are cooked, etc
> for your IHC slides, for your special stains slides, and even for your H
> slides.
> I would welcome your suggestions and feedback.
>
> Thank you so much!!!
>
> Deanne Knutson
> Supervisor
> Anatomic Pathology
>
>  [X]
>  "Let All Be Received as Christ."
>
>
>
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

Re: [Histonet] [EXTERNAL] Deplastifying MMA

[Dorothy Hu via Histonet]

Hi Lori,

Would you please tell me which company and catalog # of  tetrahydrofuran
you used before?
For how many minutes you did for deplastifying in tetrahydrofuran?
Thanks in advance.

Dorothy

Message: 3
Date: Wed, 14 Feb 2018 20:05:16 +
From: "Garcia, Lori, M.Sc." 
To: "reuel.corne...@tsrh.org" 
Cc: "histonet@lists.utsouthwestern.edu"

Subject:
Message-ID:


Content-Type: text/plain; charset="us-ascii"

Hi Reuel,

One thing you could try is THF (tetrahydrofuran). We have used it in the
past to rapidly dissolve the MMA on slides.

Good luck,
Lori
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

[Histonet] Deplastifying MMA

[Dorothy Hu via Histonet]

>
> Hi  Reuel
>

We had the same thing as you. It was caused by bad MMA, I tested. Write
down the lot # and ask refund. Test new lot # 1st of MMA in the future. It
was very frustratingI know.
Best luck.

Dorothy





> Message: 2
> Date: Mon, 12 Feb 2018 21:07:15 +
> From: Reuel Cornelia 
> To: "histonet@lists.utsouthwestern.edu"
> 
> Subject: [Histonet] Deplastifying MMA
> Message-ID:
>  namprd17.prod.outlook.com>
>
> Content-Type: text/plain; charset="iso-8859-1"
>
> Hello histonetters,
>
> I have a problem with my MMA plastic section on my slide. I could not
> remove the plastic MMA even if I leave the section slides in Xylene for
> several days with heat incubation at 60 degrees. This is the first time
> that happened to my section for several years of doing the same thing over.
> I was thinking that it was my Xylene lot number so I tried to change it but
> still does not remove the plastic, and then Itried different solvent
> Acetone, and Ethylene glycol Monoethyl ether but still it does not work.
> Can I ask for help if anyone knows what was going on and what would be the
> best way to remove this plastic from my section?
>
> Just to give you my embedding solution was MMA -94% , dibutly phthalate-
> 5% and perkadox 16- 0.5%. This have been my solution for years and I do not
> have any problem with the removal of plastic section until now. I was
> thinking that my MMA (M55909) different lot number from Sigma Aldrich may
> have cause this because even I tried to use the MMA to dissolve my plastic
> it does not work.
>
>
> Thank you for and any opinions or protocols are greatly appreciated.
>
>
> Reuel Cornelia
>
> TSRH
>
> 214-559-7766
>
>
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

[Histonet] EDTA decalcification tissue issues

[Dorothy Hu via Histonet]

We didn't compare EDTA with formic acid. But heard that formic acid also
can do many IHC and ISH, if not all the antibodies and all probes. I always
have problem of bone falling off, no matter what kind of slides. Thinking
both PFA and EDTA affect on this issue. So trying frozen tape unfixed,
undecalcified bone now. If anyone has research paper regarding this, please
share.
Thanks.

Dorothy Hu
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

[Histonet] Cut FFPE Slides for ISH

[Dorothy Hu via Histonet]

Hi Jennifer,

My ISH slides (FFPE) were stored in -20oC for several months or more than
one year and the targets were less abundant. I didn't use RNAscope though,
which is more sensitive. I don't know what kind of RNA your researcher try
to locate and need to do ISH within 24 hours after cutting?  Fresh cut
section will yield more signal for sure. I think as long as you have a few
copies of genes in your tissue, RNAscope should be able to detect.

Dorothy Hu
HSDM







> Message: 2
> Date: Mon, 28 Aug 2017 15:30:08 +
> From: "Jennifer  Phinney" 
> To: "Histonet@lists.utsouthwestern.edu"
> 
> Subject: [Histonet] Cut FFPE Slides for ISH
> Message-ID:
>  namprd05.prod.outlook.com>
>
> Content-Type: text/plain; charset="us-ascii"
>
> Hello Histonet,
> I am hoping someone can point me in the right direction to find some
> references for how long FFPE slides intended for ISH can be kept before
> staining.  I have a researcher who swears their slides have to be stained
> within 24 hours of being cut, which does not sound correct to me.  I've
> used ACD's RNAscope before and their protocol lists the timeframe as 3
> months (so this is a bit of a difference).
>
> There's a lot of information about fixation times, but I've come up empty
> handed for cut slides.
>
> Thanks everyone!
>
> Jennifer Phinney QIHCCM
> Project Coordinator
> Kansas State University
> Veterinary Diagnostic Lab
>
>
>
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

[Histonet] KOH softener to "decalcify" bone?

[Dorothy Hu via Histonet]

I know someone used KOH with EDTA together to decalcify bone.
It is worthwhile to try KOH only to soft bone and kept dynamic labeling.
If anyone had tried successfully, please let us know.
Thanks.


>
> Today's Topics:
>
>1. Fabric softener to "decalcify" bone? (Angela Lamberth)
>2. Re: Fabric softener to "decalcify" bone? - Downy has  formic
>   acid?? (Angela Lamberth)
>
>
>
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

Re: [Histonet] Histonet Digest, Vol 157, Issue 13 Kawamoto's film method (Jamie McGinness)

[Dorothy Hu via Histonet]

Please check this web for the answer of your question.
http://section-lab.jp/English/Referece%20E.htm

Dorothy Hu
>
> 
> I am looking into trying the Kawamoto's film method. ?I have the order sheet 
> for the cryofilm sheets and other supplies. ?I did have a few questions.
> 1. ?I was wondering if anyone knows when you order a sheet how many slides 
> can be produced from that sheet? ?2. ?Does anyone know what the transfer film 
> is??3. ?What are the advantages to using their SCCM mounting medium ?and the 
> difference between the four types offered G1, R1, R2, R3?4. ?What are the 
> advantages of using heir SCEM embedding medium and the difference between the 
> SCEM and SCEM L1? ?5. ?Does anyone know the differences of the cryofilm 
> 2C(9), 2C(10), and 3C(16UF)?
> Thanks so much for any information you .
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

Re: [Histonet] cryosectioning undecalcified samples (Caroline Miller)

[Dorothy Hu via Histonet]

Hi,

I compared cryojane with Kawamoto's method. I prefer the later. Please see
the following paper.


Arch Histol Cytol.  2003
May;66(2):123-43.
Use of a new adhesive film for the preparation of multi-purpose
fresh-frozen sections from hard tissues, whole-animals, insects and plants.
Kawamoto T

1.

Good luck.

Dorothy
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

Re: [Histonet] Histonet Digest, Vol 147, Issue 21"............Double stain IHC question

[Dorothy Hu via Histonet]

I wonder if streptavidin Alexa (495 or 488) can be use to bind to biotinlyted 
primary. So no worry about species issue? May need to do biotin blocking first 
to get ride of endogenic biotin I guess. 

Dorothy Hu


Sent from my iPad

> On Feb 20, 2016, at 1:00 PM, histonet-requ...@lists.utsouthwestern.edu wrote:
> 
> Double stain IHC question

___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

[Histonet] Decalcification with formic acid sodium citrate

[Dorothy Hu via Histonet]

There was a paper 
http://www.genedetect.com/Merchant2/ExampleRefs/Decalcifying_protocols.pdf
Talking about formic acid (Morse solution) can get as good result as EDTA in 
ISH. 
FYI.

Dorothy Hu


 Today's Topics:
 
   1. Re.  Decalcification with formic acid sodium citrate
  (Gayle Callis)
   2. Re: Re. Decalcification with formic acid 
 
 Merissa and Tim,  
 
 
 
 This formic acid decalcifying solution is basically the classic Evans and
 Krajian fluid (Sheehan and Hrapchak,   Theory and Practice of
 Histotechnology, 2nd edition, P.92).  Shandon has added other ingredients
 for some reason, and has kept those concentrations proprietary.   You really
 don't need to add a surfactant or PVP emulsifier when making up this
 decalcifying agent.   Simply use the classic recipe for successful
 decalcification.   This is also referred to as buffered formic acid and in
 some publications an acidic buffer.  It is excellent if IHC is needed and
 less damaging, obviously, than a strong mineral HCL acid decalcifiers.  
 
 
 
 Sodium citrate crystals (a buffering salt) 10 g 
 
 90% formic acid stock25 ml  
 
 Distilled water75 ml   
 
 
 
 One can calculate the concentration of formic acid i.e. approx. 4.5% since
 is it made from 90% formic acid stock.  
 
 
 
 Don't bother with the surfactants or PVP.  
 
 
 
 Enjoy an excellent in house formic acid decalcifying solution.  I also
 suggest you read Sheehan and Hrapchak textbook chapter on bone as a way to
 familiarize yourself with decalcifiying solutions that manufacturers now
 supply with some modifications.  Some manufacturers will refer to these
 methods but probably prefer not to do this since they want you to buy their
 commercial product that is obviously a time saver with elimination of having
 to store stock acid solutions.   The classic methods made in house are
 excellent if you have time to make them up.   Formic acid with sodium
 formate is another popular buffered formic acid.   I suggest you look for
 another source/manufacturer of the your favorite decalcifier in question as
 more than one company will make it.  Decal Corp, recently sold to Stat Lab,
 could also be the source as Shandon isn't the only game in town.   Others
 are Newcomer Supply, Poly Scientific.  Not having to make it up may remain
 your preference. 
 
 
 
 Gayle M. Callis 
 
 HTL/HT/MT(ASCP) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 Written by Tim and Merissa:   
 
 
 
 Merissa,
 
 
 
 Water77-80   solvent
 
Formic acid  21-23   active ingredient
 
 Fluorad  1   surfactant  - a
 wetting agent to make the solution wet the bone more easily
 
Sodium citrate   1   emulsifier , buffer
 
 Polyvinyl pyrrolidone1   emulsifier 
 
 
 
 They say less than one percent of the last three, but you really have no
 idea whether that is 1%, .1% or .01%. It could be any of those.
 
 
 
 But all those surfactants and emulsifiers are meant to keep the solution
 viable for long periods on the shelf. When you make it fresh you don't
 really need them.
 
 
 
 You can either buy a different decalcifier, or make your own. Making your
 own with just the water and acid will work just fine. 
 
 
 
 
 
 Tim Morken
 
 Pathology Site Manager, Parnassus 
 
 Supervisor, Electron Microscopy/Neuromuscular Special Studies
 
 Department of Pathology
 
 UC San Francisco Medical Center
 
 
 
 -Original Message-
 
 From: M.O. via Histonet [mailto:
 http://lists.utsouthwestern.edu/mailman/listinfo/histonet histonet at
 lists.utsouthwestern.edu] 
 
 Sent: Wednesday, July 22, 2015 1:24 PM
 
 To:  http://lists.utsouthwestern.edu/mailman/listinfo/histonet histonet at
 lists.utsouthwestern.edu
 
 Subject: [Histonet] understanding reagents in decalcifier; making it
 in-house
 
 
 
 Hello Histonet
 
 
 
 The supplier for our decalcifier, TBD-2 from Shandon, is having issues with
 getting the product out and we will not be receiving it for at least another
 month.  Our samples are piling up and I don't know what I should do, but
 maybe I can make the decalcifier in-house.  I am wondering if I can make my
 own based on the reagents they listed and their percentages and if certain
 reagents are not actually necessary.
 
 
 
 The samples we typically decalcify are mouse knees (decal time = 2 days),
 mouse spines (3 days), human bone slabs about 7mm in thickness (7-12 days).
 Fixation is in zinc buffered formalin, then decalcification, then 70% EtOH.
 Our choice to use TBD-2 is due to the gentle decalcification for IHC and we
 get GREAT results.
 
 
 
 Composition of Shandon TBD-2 Decalcifier:
 
 ComponentWeight %
 
 Water  77-80
 
 Formic acid 21-23
 
Fluorad   1
 
 Sodium citrate   1
 
 Polyvinyl pyrrolidone