[Histonet] Histonet Digest, Vol 195, Issue 6

Sun Feb 9 22:31:10 CST 2020

Dear All,
I am planning to introduce RNAscope in my pathology core facility, we are currently gathering information on both chromogen detection and florescent detection, but the company we are looking into for reagent ordering which is ACDbio has a wide range of channel 1  and channel 2 probes of human species, but limited channel 3 and channel 4 probes, they say you can even custom  design your probes but they are expensive.

Can anyone refer What detection system are you using or would recommend and why?

Regards
Karim
Research Associate
Aga Khan University Hospital
Karachi

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Sent: Sunday, February 9, 2020 11:00 PM
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Subject: Histonet Digest, Vol 195, Issue 6

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Today's Topics:

   1. Re: How to Reduce Tissue Autofluorescence (Hobbs, Carl)
   2. Re: How to Reduce Tissue Autofluorescence (John Kiernan)

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Message: 1
Date: Sat, 8 Feb 2020 18:56:09 +0000
From: "Hobbs, Carl" <carl.hobbs at kcl.ac.uk>
To: histonet <histonet at lists.utsouthwestern.edu>
Subject: Re: [Histonet] How to Reduce Tissue Autofluorescence
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<AM6PR03MB453481EF5BB4869F146FCEA7C41F0 at AM6PR03MB4534.eurprd03.prod.outlook.com>

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Hi
Do you refer to FIF?
Or...autofluorescence?
Different...as you prob know so, apologies in advance.
The Wright Cell imaging Facility ( Toronto western research Inst) pdf: very informative
FIF: I have tried Glycine, Ammonium chloride and Na-borohyd.
I got best results with Glycine
However....none are great.
Multi spectral imaging is the best way forwards but....expensive.
Sure, in Lambda mode using Zeiss Zen on a confocal gives good results ( tho I have forgotten how to do it, sigh) After setting up you hit the fluorescence you don't want...hit the one you want....if the wavelengths are different, what you don't want you eliminate, electronically.
Vectorlabs sell an excellent kit for FIF elimination...sure over-expensive, given the ingredients.
We found that dilution of their working reagent by x2- x4 gave best results As good as multispectral imaging, imho.
Autofl of lipofuscin is supressed using Sudan BlackB.

Good luck

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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Message: 2
Date: Sun, 9 Feb 2020 05:55:27 +0000
From: John Kiernan <jkiernan at uwo.ca>
To: "histonet at lists.utsouthwestern.edu"
<histonet at lists.utsouthwestern.edu>,Arun Jyothi S.P
<arunjyothisp at gmail.com>
Subject: Re: [Histonet] How to Reduce Tissue Autofluorescence
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There's a very brief article (downloadable PDF) from 2002 about suppressing autofluorescence, with a few references, at https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.researchgate.net%2Fpublication%2F10971457_Suppressing_autofluorescence&data=02%7C01%7Ckarim.ruknuddin%40aku.edu%7C9a6dffb091094523889608d7ad8a0353%7Ca5d4252a02f94e6096f09733baae4919%7C0%7C0%7C637168680646861537&sdata=gT64wOQByicxQn6OF9%2Fnu4A8rylTvcWFfYul736i7kA%3D&reserved=0
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(PDF) Suppressing autofluorescence - ResearchGate<https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.researchgate.net%2Fpublication%2F10971457_Suppressing_autofluorescence&data=02%7C01%7Ckarim.ruknuddin%40aku.edu%7C9a6dffb091094523889608d7ad8a0353%7Ca5d4252a02f94e6096f09733baae4919%7C0%7C0%7C637168680646871533&sdata=ZqaGFWGMm2BVRrlwdW1DYxsk5Ozkym63d2MGAMhdvXY%3D&reserved=0>
A 'read' is counted each time someone views a publication summary (such as the title, abstract, and list of authors), clicks on a figure, or views or downloads the full-text.
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This PDF file also has short peer-reviewed histotechnical tips on 5 other topics. Fun for all there.

For something more recent on autofluorescence, try:
Davis AS, Richter A, Becker S, Moyer JE, Sandouk A, Skinner J, Taubenberger JK (2014) Characterizing and diminishing autofluorescence in formalin-fixed paraffin-embedded human respiratory tissue. J. Histochem. Cytochem. 62: 405-423.
They compared 9 procedures and favoured 3: sodium borohydride, Sudan black B and another dye called eriochrome black T. The last-named dye is CI 14645, Mordant black 11, a monoazo  dye very briefly described on page 108 in Conn's 9th edn (1977) with the preferred name chromogen black ETOO; it's not in Conn's 10th edn (2002). Sodium borohydride reacts with aldehydes and probably reduces fixative-induced fluorescence of proteins and the native fluorescence of lipofuscins. The black dyes may work by absorbing more weakly emitted light. Sudan black B can stain lipofuscin black even in in paraffin sections.

Using ?home brew? reagents is always the best way to go, because you can avoid buying simple products sold at high prices with fancy names. Avoid trying anyone's unexplained "working protocol" because annotated pieces of paper get passed along in labs and can induce well educated people to do things that are obviously wrong.

It is necessary to know the reason for each step in a lab procedure. You identify as a research assistant, so you must have a boss. Probably your boss should be online along with you, asking histonetters for advice about reducing autofluorescence.

That's quite enough from me, on 9 Feb 2020.
John Kiernan  (Anatomy, UWO, London, Canada) = = = ________________________________
From: Arun Jyothi S.P via Histonet <histonet at lists.utsouthwestern.edu>
Sent: 06 February 2020 10:18
To: histonet at lists.utsouthwestern.edu <histonet at lists.utsouthwestern.edu>
Subject: [Histonet] How to Reduce Tissue Autofluorescence

Dear All,

Kindly share your working protocol using  ?home brew? reagents to reduce tissue auto-fluorescence.

Thank you
Arun Jyothi S.P.
Research Assistant
Cancer Research
RGCB
Trivandrum
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