Re: [Histonet] IHC DAB

[Ana Maluenda via Histonet]

Hi Kristy,


As the others said, so many things that could be on the way...But making sure 
your system is based on a HRP enzyme is definitely a good start, as mentioned 
by Joe.


If you can share details, I might be able to help too.


Welcome to the IHC world!


Cheers,


Ana


Ana Maluenda
Research Assistant/Laboratory Manager
Atherothrombosis and Vascular Biology Laboratory



From: Joe Myers 
Sent: Saturday, 2 May 2020 4:49 AM
To: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] IHC DAB

Kristy:
I’m thinking along the same lines as Paula; is it possible that your detection 
reagent contains only Alkaline Phosphatase  (ALP) as there a reactive enzyme?  
If so, a peroxide/DAB solution simply won’t react with it.  Can’t wait to see 
your protocol, with detailed descriptions of the antigen, pretreatment reagent 
and ‘labeling’ enzyme.
Cheers,
Joe Myers, M.S.,CT/QIHC(ASCP)




From: Kristy Castillo via Histonet

 
Sent: Friday, May 1, 2020 11:00 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] IHC DAB

Hi Histonetters,
We are starting our IHC (fun times), we are having trouble with the DAB 
lighting up.  Processed for 5 thru 10 minutes and still nothing.  Our permanent 
red is working just fine.  Any ideas.
Thank you!
Kristy
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Re: [Histonet] Pregnant in histo lab. Am I safe?

[Ana Maluenda via Histonet]

Hi Valerie,

I don't know how it is in US, but in Australia what I did was to liaise with 
our Occupational Health and Safety Department to check which measurements 
they'd recommend about having pregnant workers around the lab.

My lab however is not heavily Histology and the tissue processing is done 
off-site, so the exposure to chemicals like Xylene and Formalin is not as much 
as probably for you as a Histotech. But regardless, they helped with general 
queries and PPE recommendations (especially fitting respirators).

Hope you find a good solution for both sides.

Kind regards,

Ana


Ana Maluenda
Research Assistant/Laboratory Manager
Atherothrombosis and Vascular Biology Laboratory

Baker Heart and Diabetes Institute
75 Commercial Road, Melbourne VIC 3004



From: Valerie Laughlin [histology...@gmail.com]
Sent: Sunday, 19 January 2020 4:38 AM
To: Jessica Phillips; histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Pregnant in histo lab. Am I safe?

Yes, going to HR might be my next step before I consider other options.
The supervisor bough a respirator with generic filters without knowing he
had to buy specific filters for xylene and formalin. We also have regular
latex gloves that are definitely not resistant to xylene. Xylene melts the
gloves away.

On Saturday, January 18, 2020, Jessica Phillips 
wrote:

> I think you need to go to HR and find out what your options are. In the
> meantime, you need a respirator, specifically with a filter for xylene and
> a separate filter for formalin. You will also need some xylene resistant
> gloves with the long cuffs.
>
> -Jess
>
> On Sat, Jan 18, 2020, 4:31 AM Valerie Laughlin via Histonet <
> histonet@lists.utsouthwestern.edu> wrote:
>
>> Hello everyone. I am currently in the last weeks of my first trimester of
>> my pregnancy.
>>
>>
>> I have asked this question to my Ob-Gyn, family and general pregnancy
>> forums but I wanted to ask people who understand the field of
>> Histotechnology better.
>>
>>
>> I have been very concerned about the side effects of the chemicals that
>> might have on my baby.  The lab works with the typical stuff
>> (formaldehyde,
>> xylene, alcohol of different percentages, glacial acetic acid, stains etc)
>> They make the fixative from scratch.
>>
>>
>> I had to inform my supervisor and manager. I didn’t get the most positive
>> reaction from them but I don’t care as this is my personal business and I
>> have rights like everybody else.
>>
>>
>> I gave them a letter from my doctor informing my pregnancy and that I
>> should be kept away from the chemicals for my own safety.
>>
>>
>> They acknowledged the letter but still decided to buy a respirator mask
>> for
>> me which is fine. It’s good to have protective equipment no matter the
>> circumstance.
>>
>>
>> I told them that I can do the same tasks I do every day such as grossing
>> but with a mask, embedding, cutting and filing but that I don’t feel
>> comfortable changing the chemicals of the tissue processor and slide
>> stainer, and mixing chemicals. Also that I can’t dump the chemicals in the
>> biohazard room as there is not enough ventilation.
>>
>>
>> Literally an hour after I informed this a nurse who was working in a rojom
>> close to the biohazard room had a negative reaction and had to be sent to
>> the ER where she was there for days. She blamed the chemicals  from the
>> biohazard room. Other nurses who work close to that room had reported
>> negative side effects as well. This situation made me more uncomfortable
>> specially when my coworkers think the nurses are over reacting and it has
>> to be some other cause because they don’t get the same reactions.
>>
>>
>> My biggest concern is that despite the letter of my doctor and what
>> ocurred
>> in the past weeks with the nurse I am still feeling pressured by my
>> coworkers to work with the chemicals as they feel that a mask, a lab coat
>> and gloves is enough protection. I am unsure about this.
>>
>>
>> I didn’t get a proper fit test for my respirator by the way. I have worked
>> for another corporation where they did that right after getting hired.
>>
>>
>> I have read that chemicals can be absorbed through the skin too.
>>
>>
>> I just want to know the opinion of pregnant  lab techs and supervisors who
>> have worked with them.
>>
>>
>> I have read older threads about this in this forum before and everybody
>> had
>> positive and negative experiences. Some workers were completely removed
>> from the lab while others kept performing the same tasks. Some say their
>> babies turned out healthy while others blame the job for causing short and
>> long term
>>
>> health issues  for the babies.
>>
>>
>> Most of the employers protected the pregnant worker from the chemicals to
>> avoid any risks which I feel that’s the direction my employer should take.
>> There are 3 other histotechs in the lab and they don’t seem happy to have
>> that extra task in their hands, despite 

Re: [Histonet] IHC mouse optomization

[Ana Maluenda via Histonet]

Hi Blanca,

It all depends on what you are trying to stain, how the tissue has been 
harvested/processed and what are your antibodies. If you are trying to use a 
primary mouse on mouse tissue, that is always tricky. You will need to probably 
explore your options with M.O.M kits. Otherwise, you may need to check your 
primary and secondaries. If you are using a secondary that is rat anti-mouse, 
you may also need to use an absorbed mouse secondary, to avoid too much 
non-specific staining.

Kind regards,

Ana

Ana Maluenda
Research Assistant/Laboratory Manager
Atherothrombosis and Vascular Biology Laboratory

Baker Heart and Diabetes Institute
75 Commercial Road, Melbourne VIC 3004
P (03) 8532 1359 E ana.malue...@baker.edu.au W www.baker.edu.au


-Original Message-
From: Margaryan, Naira 
Sent: Friday, 14 June 2019 3:27 AM
To: Blanca Lopez ; 
histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] IHC mouse optomization

Biocare Medical has pure ready to use secondary..

Naira

-Original Message-
From: Blanca Lopez 
Sent: Wednesday, June 12, 2019 10:57 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] IHC mouse optomization

Morning,
I need a good advice in optimizing Mouse antibodies for research purposes. I 
use Dako/Agilent products. COX6B2 from Sigma is giving us hard times. Dako kit 
HRP has a mixture of mouse and rabbit so we think that might be the reason 
become negative.
Is there any tips for optimizing antibodies special for mouse tissue? Or if you 
can share your best procedures in IHC mouse stains. I can have a different ways 
and products to try. Any suggestion or opinions count. If you have any website 
that I can learn more about IHC for research mainly done in  Xenograft.
Thank you everybody:)

Blanca Lopez HT (ASCP)cm
Senior Histotechnologist
UT Southwestern Medical Center
Harold C. Simmons Comprehensive Cancer Center UTSTR Biorepository Tissue Lab
6000 Harry Hines Blvd NB5.102
Dallas, Texas 75390
214-648-7598
blanca.lo...@utsouthwestern.edu





UT Southwestern


Medical Center



The future of medicine, today.




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[Histonet] Platelet staining on FFPE mouse tissues

[Ana Maluenda via Histonet]

Hi everyone,

Does anyone has experience performing IHC staining for platelets on mouse FFPE?

I've been trying for a while with different primary antibodies unsuccessfully. 
I found two that work in cryosections, but not a single hint of positivity on 
FFPE tissues. The samples have been prepared as usual (NBF fixation, graded 
EtOH, Xylene, wax...) and thin sections were collected by usual procedures 
(think they are 5 um). Unfortunately I have limited resources, so my protocol 
has been manual staining (no autostainer) and I have only microwave or water 
baths available around to play with Ag retrievals. I also already tried Sodium 
Citrate and Citrate buffers at pH = 6 and 9. Anyone has a protocol that works 
and that they would be willing to share?

Much appreciated!

Kind regards,

Ana
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Re: [Histonet] Unstained slides

[Ana Maluenda via Histonet]

Hi all,

It has been very interesting reading all your comments on unstained slides. 
This has been a forever discussion I always have everywhere I go in different 
research institutes. So expanding the topic, I wonder what's everyone's opinion 
on unstained cryosections? How long are they reliable to be used for IHC?

Kind regards,

Ana

Ana Maluenda
Research Assistant
Atherothrombosis and Vascular Biology Laboratory

Baker Heart and Diabetes Institute
75 Commercial Road, Melbourne VIC 3004
P (03) 8532 1359 E ana.malue...@baker.edu.au W www.baker.edu.au


-Original Message-
From: Hobbs, Carl [mailto:carl.ho...@kcl.ac.uk]
Sent: Tuesday, 4 September 2018 4:54 AM
To: histonet 
Subject: Re: [Histonet] Unstained slides

I agree: cut only the sections needed.
Saves space.
Sure, you lose several sections of tissue when cutting more sections.
That is acceptable because, if this "oxidation" theory is true, then the 
initial sections will be no good.
However, careful organisation of exptl procedure before actual cutting will 
work very well.

Actually, not many Ags get "oxidised"for eg: I can demonstrate GFAP in 
sections that are a year old ( sure, they are stored at 4C just in case) These 
slides are used for Yr 1 BSc practicals and are consistently positive.
Nobody knows why some Ags ( and not others) lose their antigenicity, imho 
Oxidation is a vague reasoning.
Just like nobody really knows why HIER works: however, I am in the dipole 
moment school of thought, rather than the Ca++ skool Sure, in Formalin-fixed 
specimens.

Curious-illy

Carl

Carl Hobbs FIBMS
Histology and Imaging Manager
Wolfson CARD
Guys Campus, London Bridge
Kings College London
London
SE1 1UL

020 7848 6813

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Re: [Histonet] IHC validation

[Ana Maluenda via Histonet]

Hi Terri,

This is actually a very valid note. This has been in my mind for years, but 
never had the chance to put in action in a research environment (I always try 
to bring to the research field the efficiency and money saving strategies we 
use in diagnostics).

Where do you usually order your unstained slides from?

Much appreciated for the advice.

Kind regards,

Ana

Ana Maluenda
Research Assistant
Atherothrombosis and Vascular Biology Laboratory

Baker Heart and Diabetes Institute
75 Commercial Road, Melbourne VIC 3004
P (03) 8532 1359 E ana.malue...@baker.edu.au W www.baker.edu.au


-Original Message-
From: Terri Braud [mailto:tbr...@holyredeemer.com]
Sent: Wednesday, 21 March 2018 4:54 AM
To: 'histonet@lists.utsouthwestern.edu' 
Subject: Re: [Histonet] IHC validation

Just another note:  You can order unstained tissue microarrays with the 
prerequisite number of cases, both positive and negative, and stain your 
validation all on one slide.  I've done this for years and for 3 different 
validations of entire IHC platform changes, ranging from 40 to over 100 
antibodies each time.  Saves time and money.

Terri L. Braud, HT(ASCP)
Anatomic Pathology Supervisor
Laboratory
Holy Redeemer Hospital
1648 Huntingdon Pike
Meadowbrook, PA 19046
ph: 215-938-3689
fax: 215-938-3874
Care, Comfort, and Heal

Message: 2
Date: Fri, 16 Mar 2018 06:54:30 -0700
From: "Paula" 
Subject: [Histonet] Antibody Validation CLIA

Hello,
We've been discussing about the quantity of slides to run as a validation for 
IHC antibodies. We are governed by CLIA, and we would like to know if there is 
a set number of slides to run for a particular antibody we would like to bring 
in-house for Validation.  I think CAP requires 20 slides..?
And so we are asking if there is  a requirement with CLIA to run a certain 
number of slides, or is it up to us (the laboratory director) to decide how 
many slides to run for Validation/Verification.
Thank you in advance
Paula



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[Histonet] Frozen section - IHC for 6x HisTag

[Ana Maluenda via Histonet]

Hi everyone!

Does anyone have experience with immunohistochemistry or immunofluorescence 
anti-6x HisTag on frozen sections, mouse tissue? I can find online lots of 
references for immunocytochemistry, but not much on tissue staining. Any help 
or hints would be much appreciated!

Kind regards,

Ana

Ana Maluenda
Research Assistant
Atherothrombosis and Vascular Biology Laboratory

Baker Heart and Diabetes Institute
75 Commercial Road, Melbourne VIC 3004
P (03) 8532 1359 E ana.malue...@baker.edu.au 
W www.baker.edu.au

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Re: [Histonet] Paraformaldehyde Fixed Tissue

[Ana Maluenda via Histonet]

Hi Sandra,

I haven't myself particularly worked with brain and spinal cord, but majority 
of my protocols in my old job used fixation in 4% PFA (24 hours at 4-8oC) and 
routinely process (or transfer to graded EtOH, if not processing immediately).
However, our routine process didn't include a first step in 10% NBF, since PFA 
plays the role of fixation. Therefore, after PFA, we would have 70% EtOH, 80% 
EtOH, 95% ETOH, 2x EtOH the Xylenes and wax [assuming you are referring to 
FFPE?].
Also mouse tissue can be small and delicate, so I remember running liver, 
spleen, kidney and thymus (soft tissues I worked with) in a short cycle 
(similar to what I would do for biopsies).

Hope this helps!

Kind regards,

Ana


Ana Maluenda
Research Assistant
Atherothrombosis and Vascular Biology Laboratory

Baker Heart and Diabetes Institute
75 Commercial Road, Melbourne VIC 3004
P (03) 8532 1359 E ana.malue...@baker.edu.au W www.baker.edu.au

-Original Message-
From: Sandra Cheasty [mailto:sandra.chea...@wisc.edu]
Sent: Tuesday, 29 August 2017 1:30 AM
To: Histonet (histonet@lists.utsouthwestern.edu) 

Subject: [Histonet] Paraformaldehyde Fixed Tissue

Hi all,
We are having difficulty sectioning mouse tissue, (brain, 
spinal cord, liver, and spleen), on paraformaldehyde fixed tissue. Has anyone 
had issues with paraformaldehyde fixed tissue? They were processed routinely, 
starting in 10% NBF, with other tissues, and we are cutting them at 3u.
Thank you!
Sandy

Sandra J. Cheasty, HT (ASCP)
Histology & Necropsy Supervisor
UW-Madison, School of Veterinary Medicine


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[Histonet] IHC for secreted proteins and cytokines in frozen sections - Pre-fix or post-fix?

[Ana Maluenda via Histonet]

Hi everyone,

Just wondering what is people's opinion and protocols for IHC in mouse frozen 
sections targeting secreted proteins and cytokines. I see lots of places using 
fresh frozen sections/snap-freeze and cold acetone or methanol/ethanol 
post-fixation. Is this an issue when it comes to diffusion of such proteins in 
the tissue, since they are not well localized or linked to cellular structures? 
Would it be better to pre-fix (either immersion in 4%PFA or infusion with 
fixative) than post-fix?

Any thoughts would be much appreciated.

Kind regards,

Ana

Ana Maluenda
Research Assistant


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[Histonet] H on frozen sections

[Ana Maluenda via Histonet]

Hi everyone,

Just wondering if anyone out there had already had issues with commercial 
Haematoxylin and Eosin. I started working in a lab that recently purchased 
Sigma Mayer's Haematoxylin (MHS-16) and 1% Alcoholic Eosin (Fronine - II016). 
We work with fresh frozen sections of mouse tissue. Since then, H protocol 
haven't worked well. I tried different protocols, following the product sheet 
as well as following other researcher's protocols and none worked. Both 
Haematoxylin and Eosin are very faint, with Eosin been uptake very 
inconsistently even on the same section (some of the areas are not even stained 
at all). I also used sections from different projects, with different people 
performing tissue harvest, OCT embedding and sectioning (so it looks more a 
problem with the staining protocol rather than the specimens) and tried 
increasing the staining times.

Any ideas would be much appreciated. Also, what are people using as commercial 
staining Haem and Eosin (brand and catalogue number)?

Thanks very much!

Kind regards,

Ana

Ana Maluenda
Research Assistant
Atherothrombosis and Vascular Biology Laboratory

Baker Heart and Diabetes Institute
75 Commercial Road, Melbourne VIC 3004
P (03) 8532 1359 E ana.malue...@baker.edu.au 
W www.baker.edu.au

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[Histonet] Anti-fibrin antibody

[Ana Maluenda via Histonet]

Dear all,

I've been trying to optimise an immunohistochemistry protocol for fibrin that 
has not been working. Was wondering if there is anyone that would have any 
recommendation of staining protocols for fibrin or recommendation of 
anti-fibrin antibodies?
We are working with cryosections, mouse tissue, embedded fresh (no 
pre-fixation). I've tried different conditions already (different fixatives, 
blocking reagents and so on). So far, the antibodies I found commercially 
available are anti-mouse fibrinogen. This IHC is to complement special 
stainings (eg Masson and PSR).

Thanks in advance.

Kind regards,

Ana


Ana Maluenda
Research Assistant
Atherothrombosis and Vascular Biology Laboratory

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