Posted by
Ke Peng on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Two-photon-microscope-questions-tp7583010p7583030.html
Dear George:
Let me first thank you for this luxury reply and it took me already some time to run through it. Very helpful and insightful.
I will:1) include the no primary control (used to do it but then stopped as we are satisfied by the specificity of the Ab. We did not analyze the very weak signal at that time though.)2) I always acquire z-series and will start to do deconvolution (will learn this from our image specialist. We are using Autoquant. This might again be a game that somehow needs golden standards.)3) I have 4 kinds of fluophores in my samples now (405, 488, 561, 640). Inspecting another channel is very likely to give some crosstalk singles ...4) Its a protein antigen and will try KD.5) Will read the interesting references.6) I particularly like the quote ''Depend on viruses to open windows''7) We generally stick with Sigma and Abcam and overall the quality of their Abs are quite good.
Many thanks for these very helpful and interesting information.
Aro
Date: Sun, 30 Nov 2014 13:01:26 -0600
From:
[hidden email]
To:
[hidden email]
CC:
[hidden email]
Subject: Re: How to define the presence/absence of an object in immunofluorescence
Hi Aro,
Intensity 2200 of feature of interest vs 2000 of background. Camera
maximum 65535. Not enough information. Also, N=1, so more cells, and
proper controls will help. If you increase exposure time 10x you might
get values of ~22,000 vs 20,000, which would help. Personally, I would:
1. acquire more data, including no primary antibody controls.
2. acquire Z-series and deconvolve (whether widefield or confocal ).
3. acquire other fluorescence channel(s) to understand how
autofluorescent your cells and/or tissue are. I routinely acquire at
least one channel that is not supposed to have any fluorophores in it
(440nm or 458 nm excitation and "Cyan FP" emission band works well for
many kinds of cellular autofluorescence).
4. If it is a protein antigen that is supposed to be synthesized by the
cells you are detecting the antigen in or on, corroborate with RNA FISH
(more on this later).
The imaging hardware we have is able to discriminate single molecules
even in brightfield - see
http://web.stanford.edu/group/blocklab/GutierrezAJP2010.pdf(a reference I am recycling from the recent single microtubules
thread). On a clean background, a few counts above the camera offset
can be enough. In or on cells that have autofluorescence, this is much
harder.
For increasing signal over background -- when using 'clean' antibodies
-- I recommend tyramide signal amplification (TSA). This is
commercially available from both Molecular
Probes/Invitrogen/LifeTech/ThermoFisher and from PerkinElmer. Perkin
Elmer is making a push with TSA for 3plex and 6plex immunofluorescence,
http://www.perkinelmer.com/catalog/category/id/opal-multiplex-tissue-stainingespecially in conjunction with their Vectra spectral imager. Good ideas
though I recommend PeroxAbolish instead of microwaves to kill
horseradish peroxidase - my former UMiami colleagues, Dr. Takahashi and
Prof. Ichii, had outstanding CD4 and CD8 TSA immunofluorescence in
their work published in
http://www.ncbi.nlm.nih.gov/pubmed/21929847(the published images do not do justice to how bright and specific
their staining was).
There is a famous quote by Arthur Kornberg, "Thou shalt Not waste clean
thinking on dirty enzymes"
http://sandwalk.blogspot.com/2007/10/biochemist-arthur-kornberg-1918-2007.htmlTo which I'll add: "do not waste time or money on stupid antibodies",
so if you are using polyclonal antibodies (not reproducible, the rabbit
is probably dead already), reagents from companies like 'Santa Crap'
that don't do quality control, and especially companies that the USDA
has cited for violating the US animal welfare act,
https://awionline.org/sites/default/files/uploads/documents/SanatCruz-1-15-0023COMPLAINTRedacted.pdf
The first two papers in what will be a series were recently published
in Journal of Histochemistry and Cytochemistry, are open access, and
provide good advice:
http://jhc.sagepub.com/content/62/10.toc Denis
G. Baskin and
Stephen
M. Hewitt
Improving
the State of the Science of Immunohistochemistry: The
Histochemical Society’s Standards of Practice
J
Histochem Cytochem October
2014 62: 691-692
doi:10.1369/0022155414538453
Free
Full Text
Free
Full (PDF)
Stephen
M. Hewitt,
Denis
G. Baskin,
Charles
W. Frevert,
William
L. Stahl,
and Eduardo
Rosa-Molinar
Controls
for Immunohistochemistry: The
Histochemical Society’s Standards of Practice for Validation of
Immunohistochemical Assays
J
Histochem Cytochem October
2014 62: 693-697
doi:10.1369/0022155414545224
Abstract
Free
Full Text
Free
Full (PDF)
For protein antigens that you expect are being made in the same
cells that you are measuring them in, the 'Central Dogma' of DNA codes
for mRNA codes for proteins, means that you could use:
1) gene knockout (more definitive) or RNAi (less definitive, but still
pretty good) to eliminate the gene or expression. ZFNs, TALENs and
CRISPR/Cas9 all work. Humin Zhao has published TALENs at $5 each
http://www.ncbi.nlm.nih.gov/pubmed/24237314 -- which is cost
competitive with the guide RNAs needed for Cas9. CRISPR Cas9 is now
over 300 references for just 2014 (with one month left), for a tool
only published in August 2012
(
http://www.ncbi.nlm.nih.gov/pubmed/22745249) -- this decade's answer
to previous revolutions in PCR and RNAi. These also intersect with
light microscopy as in, for entry points,
http://www.ncbi.nlm.nih.gov/pubmed/25307933
http://www.ncbi.nlm.nih.gov/pubmed/24556431http://works.bepress.com/gmcnamara/63http://works.bepress.com/gmcnamara/422. RNA detection
a. in live cells by molecular beacons, or SmartFlares (commercial
name from EMD Millipore) = NanoFlares (academic name,
http://www.ncbi.nlm.nih.gov/pubmed/25404304 and
http://www.ncbi.nlm.nih.gov/pubmed/18034495).
b. single molecule RNA FISH, for examples:
Stellaris FISH ...
http://stellarisfish.smugmug.com ... one of
the galleries is a bit of a tease with respect to new hardware,
http://stellarisfish.smugmug.com/StellarVision/
RNAscope
QuantiGene
http://www.panomics.com/products/gene-expression/single-plex-assay/overview RNAscope and QuantiGene are the same branched DNA technology, from
two different vendors.
I've been doing Stellaris FISH experiments for the past several months,
and posted some of the data online, for example
http://works.bepress.com/gmcnamara/64/
George
p.s. All of Kornberg's commandments at
http://sandwalk.blogspot.com/2007/10/biochemist-arthur-kornberg-1918-2007.htmlBiochemists
also know him for creating the Ten Commandments of Enzymology. Unlike
the author of the original ten commandments, Kornberg was able to
modify and amend his commandments as new developments came along
(Kornberg, 2003).
Thou
shalt…
I.
Rely on enzymology to resolve and reconstitute biologic events
II.
Trust the universality of biochemistry and the power of microbiology
III.
Not believe something just because you can explain it
IV.
Not waste clean thinking on dirty enzymes
V.
Not waste clean enzymes on dirty substrates
VI.
Use genetics and genomics
VII.
Be aware that cells are molecularly crowded
VIII.
Depend on viruses to open windows
IX.
Remain mindful of the power of radioactive tracers
X.
Employ enzymes as unique reagents
My email signature line used
to be:
"Old Soldier's never die, they just fade away" - Douglas Macarthur.
"Old antibodies die, please throw them away" - GM.
On 11/30/2014 11:55 AM, PengKe wrote:
Dear lister:
I'm recently puzzled by a question that I felt might have puzzled some of you as well and I would like to receive some suggestions and opinions from you.
Question: In immunofluorescence analysis, sometimes we detected objects that showed very weak signals, so how can we decide whether the signal is really there or not?
To make the question a little more specific, I will give some artificial numbers. Let's say the background has the signal intensity of 2000 and the potential object showed a signal intensity of 2200. The saturating intensity of the camera is 65535. In this case can one claim the 2200 signal represent a real object?
A related question might be: is there a golden standard about how much higher a signal intensity needs to be above the background to be defined as an object?
I'm looking forward and I'll be very grateful to your opinions.
Best wishes,
Aro
--
George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales
http://works.bepress.com/gmcnamara/42