[Histonet] AFB Stain

[Adesupo, Adesuyi (Banjo) via Histonet]

Hi,
I want to know whether it is compulsory to use two controls (positive & 
negative) for AFB Stain.

Thanks,
Banjo Adesuyi
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Re: [Histonet] AFB Control Question

[Mayer,Toysha N via Histonet]

Pam,
I worked with our microbiology department and had them to obtain some positive 
organisms and with our surgery and morgue for fresh tissue.  We then inoculate 
the tissue and process it.  Positive blocks are kept on hand and used.  We also 
pass a copy of the slides back to the microbiology department to keep on file 
as a good batch.  It seems to work for us in the educational setting.  We have 
done afb, gms, and gram.  Our next batch will include Cryptococcus, and h. 
pylori.
Hope that helps!

Sincerely,

Toysha N. Mayer, D.H.Sc., MBA, HT (ASCP)
Instructor/Education Coordinator
Program in Histotechnology
School of Health Professions
UT M.D. Anderson Cancer Center
713.563-3481
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Message: 2
Date: Wed, 15 Feb 2017 15:53:07 +
From: "Marcum, Pamela A" 
To: "histonet@lists.utsouthwestern.edu"

Subject: [Histonet] AFB Control Question
Message-ID: <8c03b2f73b8e4e5593bf93faa4069...@mail13m2n1.ad.uams.edu>
Content-Type: text/plain; charset="us-ascii"

Good morning,

We are attempting to find AFB controls from human sources.  We would trade if 
we have something you need and we have available.  Does anyone have blocks they 
might share or trade without upsetting our HIPPA people on either side.  I am 
really not interested in buying slides as we cut larger blocks down and have 
controls on with the patient tissue.  Also the control slides are general in 
the middle of the slide and force to use two slides for every test (too many 
ordering and all want a control) which makes the cost prohibitive.  Our 
pathologists prefer human tissue and most of the companies I talk to either 
don't know or won't say it is animal origin or human.


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[Histonet] AFB Control

[Forest Blankenship via Histonet]

Hello Pam,

It is always recommended to have AFB control and patient tissues run separately 
in parallel to avoid contamination from the control to the patient tissue. The 
most economical way to do this is to use a staining tray and use a pipette to 
do all the steps involved then it is impossible for the bacteria to transfer to 
the patient slide.

Thank you,
Forest K. Blankenship, HTL(ASCP)
Histology Manager
Driscoll Childrens Hospital
Corpus Christi, TX

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[Histonet] Acetone freeze substitution vs fixation, and other Cryo questions

[Evan Chiswick via Histonet]

Hello Histonet members,

I am fairly new to the world of histology, but I have found this listserve to 
be very helpful.

I have a few questions which will be followed by a general description of my 
project/protocol, in case that is helpful for answering.


Question: I have read here and elsewhere of acetone used as a fixative after 
first drying the section, but other fixatives are used when acetone is used for 
freeze substitution(e.g. http://jcb.rupress.org/content/jcb/4/5/593.full.pdf). 
Does freeze substitution with cold acetone also fix the section? I want to 
avoid air drying the section as I have the impression that will distort the 
morphology.

What happens to freeze-substituted sections when I subsequently immerse then in 
staining buffer (.e.g. PBS w/BSA & Triton-x). Would that reverse the 
substitution/fixation?

If I want to subsequently store my stained slides for future use/reference, how 
should I do that? How should freeze substituted slides be stored (pre-staining)



Brief description of project.

Use highly multiplexed immunofluorescent staining of uterine sections to 
measure cellular composition, protease activity (amenable to acetone/frozen 
sections), as well as various other molecular features. Through computational 
methods, these features will be modeled in conjunction with the spatial and 
morphological features of the section. Hence, I want a tissue preservation 
method that preserves enzymatic activity (freezing) and morphological detail 
(freeze substitution/no air drying??). I am under the impression that 
air-drying is necessary to ensure attachment of sections to slide. Would the 
cryojane tape-transfer technique get me around the need for air drying?



Brief outline of protocol

Snap freeze tissue blocks in isopentane chilled by liquid nitrogen.

Section 4um with cryojane, freeze substitute with cold acetone, and store at 
-80 or liquid nitrogen.

Block sections (PBS/BSA Triton-x100, or something similar).

Stain with panel of antibodies with unique linkers attached. Wash.

Light PFA fixation (~10min) to crosslink antibodies.

Iteratively image 3-4 antigens at a time, with cyclic detection incubations, 
image, dehybridize detection, repeat).

Store slide for future use (e.g. additional immunostains, or H) (how to 
store?)



Thank you for your time.

Sincerely,

~Evan

Evan L. Chiswick
Postdoctoral Research Associate
---
Department of Biological Engineering
Massachusetts Institute of Technology
Cambridge, MA


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[Histonet] AFB Control Question

[Marcum, Pamela A via Histonet]

Good morning,

We are attempting to find AFB controls from human sources.  We would trade if 
we have something you need and we have available.  Does anyone have blocks they 
might share or trade without upsetting our HIPPA people on either side.  I am 
really not interested in buying slides as we cut larger blocks down and have 
controls on with the patient tissue.  Also the control slides are general in 
the middle of the slide and force to use two slides for every test (too many 
ordering and all want a control) which makes the cost prohibitive.  Our 
pathologists prefer human tissue and most of the companies I talk to either 
don't know or won't say it is animal origin or human.


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