[Histonet] Delays in tissue embedding after mouse tissue processing

[Erickson, Jamie E via Histonet]

Hello All,
I have a question about effects on tissues if samples are processed but NOT 
embedding immediately after processing.
We deal with mostly mouse tissues and sometime samples will process over the 
weekend and come off on a day that no one can embed straight away.
Samples are taken out of the processor and let sit on the bench and the 
paraffin coated cassette tissues harden. This delay can be hours or a few days 
if it's a weekend/ holiday.  The samples are then placed in molten paraffin to 
melt and are  embedded.
We have done this with minimal problems but are wondering if some tissues like 
skin mouse ears  could be more difficult due to this delay. Have other lab done 
this delay or have seen any problems

Thank you for any though on this sticky situation...Hehe

Jamie

Jamie ericKson, MS
Scientist II
AbbVie Bioresearch Center, Inc.
Pharmacology/Pathology
100 Research Drive
Worcester, MA 01605
OFFICE+1 508-688-3134
EMAIL  jamie.erick...@abbvie.com
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[Histonet] Kawamoto protoco

[Erickson, Jamie E via Histonet]

Hello histo-neters,
   I was able to purchase this system many 
years back from Dr. Kawamoto because we had many mouse bones we wanted to do 
IHC, ISH etc. on. After watching the video it was clear that this was not going 
to be fast and easy. Our impressions were that this was going to take a lot of 
effort, time and the investigators did not move forward with testing out the 
kit. We use the Kawamoto tape in some situations as it can give good sections 
but we did not make this a standard SOP.
   If I remember a few issues we had were that the staining was done 
completely on the sectioned tape (floating on flowing water) then the tape was 
mounted to a glass slide under a coverslip. As with everything the devil is in 
the details and there were a lot of details, from embedding samples with the 
mounting media exactly  as stated in protocol to sectioning with D-profile 
blades or disposable but  these had to be special (thicker) blades with a 
special blade holder angled if I'm remembering right.

We  modified things and made a method that worked good enough but nothing like 
the slides he sends with the kit , they are perfect.  He is truly a master of 
this craft.
His protocol may have changed from  ~ 2009 but we thought it was going to take 
a lot of training and time which is in short supply in industry.

I still like fix/ decal (formic acid or EDTA) and sucrose protect I think this 
give me good results and great sections when sectioned with tape transfer..

These are my thoughts I hope it helps,

Jamie ericKson, MS
Scientist II
AbbVie Bioresearch Center, Inc.
100 Research Drive
Worcester, MA 01605
OFFICE+1 508-688-3134
EMAIL  jamie.erick...@abbvie.com

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that any other dissemination, distribution, use or copying of this 
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should notify the sender immediately by telephone or by return e-mail and 
delete it from his or her computer.


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[Histonet] CD3 antibody for mouse FFPE

[Erickson, Jamie E via Histonet]

Hi Brett,
We run the Leica BOND RX here and we love the CD3 from Thermo  CD3 (Clone SP7)  
Rabbit Monoclonal Antibody
Cat. #RM-9107-S0, -S1, or -S (0.1ml, 0.5ml, or 1.0ml Supernatant).
We use the H2-20 retrieval which is HIER (EDTA) and it works on human , mouse 
and Rat.
It is a Supernatant so you'll have to call for the ~ concentration it is 
~160ug/ml and works at 0.8ug/ml. With the anti-rabbit polymer detection it 
looks great and the incubated time we use is only 15min.  Another good thing is 
we do not need to use a protein block which saves time.
It looks great as a double with  IBA-1 Alk phos and CD3 black (biocare Deep 
space black) and a light methyl green counter stain.

Hope that helps,
Jamie Erickson, MS
Scientist
AbbVie Bioresearch Center, Inc.
100 Research Drive
Worcester, MA 01605
Ph: 508-688-3134

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Re: [Histonet] C3 IF staining of FFPE tissue (Kristyn Ferber)

[Erickson, Jamie E via Histonet]

Hello, Kristyn,
 I don't know what species you work with but I work 
in mouse (mouse lupus kidneys) and had good luck with Dakos C3c antibody F0201 
using Dakos Proteinase K 10 minutes at room temperature.  I used a unlabeled 
version that is not available anymore but F0201 (FITC) one is said to be of the 
same clone. I had aliquot lots of the old antibody that just ran out so  I have 
not tried this FITC one yet.  If you want  to  stain using DAB I've used a 
Rabbit anti-FITC (Invitrogen) and then come back with  Anti-rabbit HRP polymer 
then DAB. Its works nice when you have a FITC primary but don't want to 
visualize with Fluorescence due to background etc..  
We have tested a lot of Ab's on paraffin for various epitopes and have a good 
success rate you just may have to test some antibodies and a few pretreatment 
conditions (EDTA, Citrate, PTK, Protease 1) we use a Leica Bond autostainer. 

Best of luck.

Jamie Erickson
Scientist
Abbvie bioresearch Center
Worcester, Ma 01605

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Sent: Monday, February 08, 2016 1:00 PM
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Today's Topics:

   1. C3 IF staining of FFPE tissue (Kristyn Ferber)
   2. Issue with Leica/Jung Histoembedder (dayuang)
   3. RELIA Histology Careers Bulletin Special Edition for  Managers
  and Supervisors - 2-8-2016 (Pam Barker)


--

Message: 1
Date: Mon, 8 Feb 2016 08:15:21 -0500
From: Kristyn Ferber 
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] C3 IF staining of FFPE tissue
Message-ID:

[Histonet] Is antigen retrieval open on the Leica BOND RX IHC

[Erickson, Jamie E]

Hi Wes,
   We have 3 bond RX units and use them for animal tissue 
staining and we try all kinds of reagents (ab, chromogens, counterstains, acid 
washes) . As far as the HEIR and Enzyme go Leica has their own HEIR (citrate  
EDTA) and an Enzyme (PTK). You can run a Dako PTK which I do at room temp or at 
37C but as far as the HEIR you are limited to their Citrate Ph= 6 and EDTA 
Ph=9, I think I'm right on the Ph's but not sure. 
Anyway, we have found that all of our Dako protocols using PT modules or 
Pressure cooker  transferred  nicely to Leica and there recommended times for 
citrate and EDTA are pretty  darn good.  It is a learning curve and there are 
Leica ways of doing things but you can  get  to do almost anything you can do 
on the bench. We are working up a lot of double now and there are so many ways 
to do it.
Hope that helps.

Jamie

JAMIE ERICKSON
Scientist II
HTL (ASCP)CM,MS


Abbvie Bioreseach Center
Pharmacology
100 Research Dr.
Worcester,Ma 01605
OFFICE    +1 508-688-3134
FAX  +1 508-793-4895
EMAIL  jamie.erick...@abbvie.com
abbvie.com

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that any other dissemination, distribution, use or copying of this 
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delete it from his or her computer.

**

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[Histonet] CD4 and CD8 mouse tumor IHC, thanks..

[Erickson, Jamie E]

Thank you to those that sent me suggestion on my CD4 problem. I have recently 
discovered the problem.
The tumors are mouse tumors but they are injected in-vivo with a test antibody 
that is Rat anti-mouse, specifically Isotype (Rat IgG2a). Hence this is why my 
secondary Rabbit anti-rat stained so much..

Next I will try to block the Rat in-vivo injected antibody with a goat anti-Rat 
secondary, as a  block prior to staining with CD4.
Or I could precomplex the CD4 with the Rabbit Anti-Rat and bind excess Anti-Rat 
with Rat Serum.

Any thoughts or suggestion on this as always would be welcomed...

Thanks again..

Jamie

Abbvie Bioresearch Center
Pharmacology
100 Research Dr.
Worcester, Ma 01605
OFFICE+1 508-688-3134
FAX  +1 508-793-4895
EMAIL  jamie.erick...@abbvie.com
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[Histonet] CD4 and CD8 background in mouse tumors frozen sections, help

[Erickson, Jamie E]

Hello Histonetters,
 First I would like to say happy 
holidays to you all, this is such a great resource to have.
Now for my question.
I'm rather vexed about this problem with staining CD4/Cd8 in mouse tumors I 
hope someone can shed light on because I'm stumped.
An investigator gave me some frozen mouse tumors and spleen to do CD4 and CD8 
stain on and I expected this to be easy...
After staining the spleens and tumor with CD4 and CD8 at (1ug/ml and 5ug/ml 
respectively) I saw the problem, the isotype control. The isotype for these 2 
Rat-mouse antibodies from BD biosciences is a Rat IgG2a.
The rat IgG2a on the mouse tumor and spleen slide were  negative and I was 
happy but when I looked at the other tumors  Rat IgG2a slide some were very 
positive. The CD4 was staining in some tumors but these tumors had background 
throughout the tissue as well. Very strange I thought, some tumors no 
background others lots of background..

As  I'm not a tumor biologist I'll just say these tumors vary in size from 
small to large,  large (1cm) and some have necrosis others do not.
So what I see is that the isotype (rat IgG2a) stained in some tumors but not 
all of them and  the spleen and a few small tumors are negative.  I only did 
Neg controls on 2 tumors one large and one small tumor with a spleen from that 
tumor bearing mouse.

I first thought peroxidase or Fc binding as I don't do a protein blocking step 
with this system, typically. I added a protein block (Dako) and increase 
peroxidase time from 5 minute to 10 but saw no difference.
It looked like my titers were high for these tumors so I titered  the antibody 
out. I  titered my CD4 down to 0.25ug/ml for these tumors  and that did not 
help the Rat IgG2a staining but the CD4 staining looked better not as dark 
brown in the spleen.
So now I need to explain my staining protocol.. We use a Leica Bond RX system 
which I use a DAB kit to detect with and it works great.

Marker (antibody, Rat Anti-mouse CD4)15
Rabbit Anti-Rat
vector lab (adsorbed to mouse)
as a linking step.  
 20
polymer (Anti-Rabbit)8
Peroxide block  
5
DAB 
  10
Hematoxylin 
 5

I next looked at the linker step
slide #1  I did a slide with no CD4 still had staining (background)
Slide #2 No CD4 and No linker No background.. cleanI think it is 
the linker..
Unfortunately I'm stuck using this linker (rabbit anti-Rat , 2ug/ml) for now..

What could be going on in these tumors ???  could I block this without changing 
my polymer (rabbit). If it is the linker then is it binding via Rabbit Fc or is 
the hypervariable region specific for something in these tumors i.e 
Anti-Rat tumor protein?
Can I pre-complex the CD4 and linker then bind up excess anti-Rat, any one done 
that???
If it is not complicated enough the linker is adsorbed to mouse.
I tried Biocares Rat-on mouse polymer but I did not see any reaction at all, 
..but I haven't investigated that fully..

Sorry for the long email ..
Hope you can help...Happy holidays.

Jamie Erickson
AbbVie BioResearch Center
Pharmacology
100 Research Drive
Worcester, MA 01605
OFFICE+1 508-688-3749
EMAIL  terry.me...@abbvie.com



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[Histonet] Section cells on a plastic trans well membrane

[Erickson, Jamie E]

Hi All,
  Has anyone tried to section cells cut from a trans well dish? 
We have cells that are cultured and attach to a trans well membrane and our 
pathologist wants to look at them on the membrane but the membrane is a thin 
plastic film...see the problem.
We fixed the cells in the trans well for 5 minutes then  cut the trans well out 
of the well and bisected it and processed overnight we use paraffin  type #1 
paraffin. The next morning we embedded it with type #9 paraffin.  The samples 
cut but  do not stay on the slide after xylene. I think the plastic does not 
adhere to the slide. Would a adhesive slide work to keep the thin plastic stuck 
to the slide??

My pathologist does not want to change to a collagen membrane  so he wants me 
to find another (harder paraffin) to use, I don't think that will help.
Has anyone does similar processing/embedding?
I'll take any help I can get..

Thanks,

Jamie
Abbvie Bioreseach Center
Pharmacology
100 Research Dr.
Worcester,Ma 01605
OFFICE+1 508-688-3134
FAX  +1 508-793-4895
EMAIL  jamie.erick...@abbvie.commailto:jamie.erick...@abbvie.com


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[Histonet] Looking for mouse antibody of LTa for use in IHC

[Erickson, Jamie E]

Hello Histonetters,

Does anyone know of a antibody to mouse LTa (Lymphotoxin alpha (TNF 
Superfamily, Member 1) (LTA)) that works in IHC.

A colleague is pulling her hair out trying various clones in formalin and 
frozen sections of mouse spleen but with no luck, please help her..

Is there one that works or is she chasing a ghost...

Thanks for any help you can provide..
Jamie

Jamie Erickson
Abbvie laboratories
Scientist II
HTL (ASCP),MS
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[Histonet] Re: Lung histology

[Erickson, Jamie E]

HI  Zakky,
   I've worked on a lot on mouse lung and the process of 
embedding can depend on what you are looking for or your pathologist opinion.
In my OVA models  I was looking at inflammation around the main stem bronchi 
and looking at goblet cell hyperplasia  using PAS stain. Because we used an 
intranasal  route of administration we wanted to look to see if there was a 
difference in PAS at the main stem bronchi compared to  distal secondary and 
tertiary bronchi so we chose to embed sagittal.
I isolated the left lobe that is the lobe with 
no accessory lobes then trimmed in  to the main stem but not to close,  this 
was done to make a flat surface for embedding. Then you can cut into the lung 
until you get a nice full or almost full length of the main stem bronchi. I did 
PAS on these and image analysis to quantitate amount of PAS production. 
Perfusion of the lungs was a must for this view.
There are lots of papers that do this by cross sections as well and stain with 
collagen deposition stains like sirus red, so you'll have to see what fits your 
study.
Whatever you choose be consistent with your location of sectioning, so you can 
compare samples across groups that are taken from the same anatomical location.

Hope that helps  Good luck,

Jamie Erickson (ASCP) HTL
Pharmacology Dept.
Abbott laboratories
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[Histonet] Staining for Osteoid in FFPE tissues

[Erickson, Jamie E]

Hello All,
Have anyone tried this for osteoid in paraffin. I understand plastic are the 
best but I can do plastics. Will this work or are there any other alternatives 
to getting osteoid in FFPE.  We use formic acid (cal-rite)  for decaling our 
process is; fix 24 Hrs then cal-rite 2 days then new cal-rite 2 days then wash 
thoroughly and process mouse Paws/tibia/vertebra.
This paper says osteoid is well differentiated by cyanuric chloride solution 
prior to decalcification (EDTA is better).
Has anyone tried this?
Jamie

IMPROVED PROCEDURE FOR HISTOLOGICAL IDENTIFICATION
OF OSTEOID MATRIX IN DECALCIFIED BONE
S. YOSHIKIT, . UENO,T . AKITA A ND M. YAMANOUCHI
Department of Oral Pathology, School of Dentishy, Showa University, 
Shinagawaku, Tokyo 142 Japan

ABSTRACT. Several improvements on the original method of Yoshiki and coworkers 
for histological
identification of osteoid matrix in decalcified bone are described in this 
report. The first, fixation of
bone with neutral buffered formalin, a popular and stable fixative, should 
produce better tissue
morphology and ensure easy handling in any laboratory. The second is a simple 
test for aged cyanuric
chloride. Aged reagents show poor or no solubility in methanol and have almost 
no effect on
differential staining of osteoid matrix. The third is an application of an 
organic acid solution in place
of neutral EDTA for bone decalcification. Reduced decalcification time with the 
acid results in rapid
preparation of bone sections. Neutral formalin fixation, immersion in the 
cyanuric chloride solution,
decalcification with an organic acid, and hematoxylin and eosin staining, all 
quite routine laboratory
procedures, yield high quality results for identification of osteoid matrix in 
bone sections.

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[Histonet] Cryosectioning question

[Erickson, Jamie E]

Hi All,
 I have a question about cryopresevation/ sectioning. I have a 
study (mouse kidneys sample)that I need to re-section the cryoblocks but the 
intern that sectioned them first did not reseal them with OCT so they have now 
seperated from the OCT (dessicated).
I can't get sections because of the tissue and OCT separate. Can I re-freeze 
them? If so how do I avoid thawing the sample. Has anyone run into this, if so 
what did you do.

   Also is there a good method for sectioning out there I want to 
improve my speed and quality of cryosections  so I hope to adopt some new 
tricks. Do people like using a camel hair  brush  or the anti-roll plate, I use 
a brush.. Is there any good refference material I should look into...

Any help is appreciated.
Thanks,

Jamie
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