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Hi Martin,

I chickened out on including (Komodo) dragon IgG in the (thought 
experiment, not 'reduced to practice') table from our immunofluorescence 
microscopy chapter (2011, book 1 chp 15 of 
https://www.cshlpress.com/default.tpl?action=full&--eqskudatarq=872&typ=ps&newtitle=Imaging%3A%20A%20Laboratory%20Manual 
... recycled from an earlier imaging book)

Tubulin               Mouse IgG1     Goat–anti-mouse IgG1 Alexa Fluor 
488 Green
Actin                   Guinea pig IgG Goat–anti-guinea pig-IgG Lucifer 
Yellow Yellow
Vimentin            Rat mAb IgM  Goat–anti-rat-IgM Cy3B Orange
Cytokeratin        Mouse IgG2a  Goat–anti-mouse IgG2a DyLight 594 (or 
Texas Red) Red
Neurofilament  Chicken IgY     Goat–anti-chicken IgY HiLyte Fluor 647 
(or Cy5) Far red
Nestin                 Rabbit IgG      Goat–anti-rabbit IgG Atto 680 
Infrared
CD31                   Donkey IgG    Goat–anti-donkey IgG IRDye800 Infrared

Quite unwieldy!

for comparison, clinical flow cytometry is now 'routinely' done with a 
lot more than 7plex direct labeled antibodies.

As for tissue section autofluorescence vs direct labeled antibody 
brightness: there are now many ways to decrease autofluorescence before 
imaging (I seem to be getting ads from Biotium once a week about their 
stuff, for example ... various authors have published using UV 
transilluminator to good and/or poor effect). There is also potential 
for new (likely expensive) fluorescence microscopes, such as Leica SP8 
FALCON (disclosure: our image core hosts FALCON workshop week of Sept 
10, 2018 ... hmmm: maybe I should measure BV421 lifetime), or other Fast 
FLIM instruments (whether upgrades or new). For examples, if NADH, 
collagen, and elastin autofluorescence lifetimes are 1 nanosecond, and 
organic dyes of similar spectra are each ~4 ns, could be 6plex just in 
blues and greens, plenty of spectra for more.

enjoy,
George


On 8/23/2018 10:33 PM, Martin Wessendorf wrote:
> *****
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> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> I'm going to take issue with George on one point: I don't think that 
> directly labeled primaries are necessarily the best way to go.  The 
> beauty of indirect immunofluorescence is that you can combine a given 
> primary antibody with any number of different fluorophores (in the 
> form of fluorophore-conjugated secondary antibodies). Once you've 
> characterized that primary antibody, you can be reasonably sure that 
> it will label the same molecule no matter what color secondary 
> antibody it's paired with (...assuming that the secondary works well 
> enough to detect any labeling!).  In contrast, each different 
> fluorophore-conjugate of a directly conjugated primary antibody, is a 
> chemically different reagent that may have different binding 
> properties and will need to be re-characterized for specificity.  
> --I.e., you would be setting yourself up for a lot of extra work if 
> you use directly conjugated primaries.  (If you've already 
> characterized the specific reagents you'll be using, then this isn't 
> an issue.)
>
> My two-cents worth!
>
> Martin Wessendorf
>
>
>
> On 8/23/2018 8:43 PM, George McNamara wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your 
>> posting.
>> *****
>>
>> As much as I am a fan of TSA (and look forward to the methyl-luminol 
>> alternative), more multiplexing options:
>>
>> https://www.ncbi.nlm.nih.gov/pubmed/29263082
>>
>> Pleiner et al 2018 JCB -A toolbox of anti-mouse and anti-rabbit IgG 
>> secondary nanobodies.
>>
>> https://www.ncbi.nlm.nih.gov/pubmed/29444803
>>
>> Commentary
>>
>> The commercial arm is www.nano-tag.com
>>
>> In many ways, nanobodies are an update of the "Pretty Poly" method of 
>> Morris and Stanley 2003
>> https://www.ncbi.nlm.nih.gov/pubmed/?term=pretty+poly+stanley
>>
>> see also Molecular Probes Zenon product line.
>>
>> These are not amplification.
>>
>> See also Brilliants and SuperBrights: ideally the fluorescence 
>> microscopy community could be using the direct labeled fluorescent 
>> antibodies routinely used in flow cytometers and FACE sorter. I 
>> mentioned to some visitors today that BD's X50 is named for their 
>> intent (and look to be on target to achieve) 50plex fluorescence flow 
>> cytometry, with several directional cell sorting (I'm spacing out on 
>> the numbers, maybe 6 way sort now, 10 way in ~3 years?). I'm also a 
>> fan of CyTOF, imaging CyTOF, MIBI-ToF, etcTOF.
>>
>> enjoy,
>> George
>> p.s. Disclosure: I came across nano-tag.com at their exhibit at 
>> Society for Neuroscience 2017, who turned me on to the Pleiner 
>> article. They were nice enough to send several free samples, which 
>> our users found worked fine on our new Leica SP8 confocal microscope 
>> (should also work fine on our Olympus FV3000RS, but that only was 
>> installed last week).
>>
>> On 8/23/2018 4:57 PM, Jeff Carmichael wrote:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> Post images on http://www.imgur.com and include the link in your 
>>> posting.
>>> *****
>>>
>>> COMMERCIAL POST
>>>
>>> In a recent conversation thread about blue fluorochromes, George 
>>> McNamara
>>> mentioned alternative labeling methods such as biotin/streptavidin and
>>> TSA-hapten.
>>>
>>> This reminded me of a customer we've been working with who has 
>>> developed
>>> what they're calling a "next generation" hapten (not TSA) labeling
>>> technology with very high specificity, affinity and amplification. 
>>> This in
>>> turn means that the primary antibody species origin doesn't matter, 
>>> and 4
>>> mouse monoclonal antibodies may be used together on the same tissue 
>>> sample,
>>> including without multiple labeling steps. The "modified" haptens are
>>> referred to as peptide bar-codes which apparently allow for significant
>>> multiplexing:
>>>
>>> https://cellidx.com/technology
>>> https://event.webcasts.com/viewer/event.jsp?ei=1187871&tp_key=9db65f5892 
>>>
>>>
>>> Chroma Technology has no commercial interest in these products per se,
>>> other than the filter sets that may be recommended for use with them.
>>>
>>> Jeff
>>>
>>> *Jeff Carmichael*
>>> *Product & Technical Marketing Manager*
>>>
>>> *[log in to unmask] <[log in to unmask]> | 802-428-2528*
>>>
>>> * <[log in to unmask]>*
>>>
>>

-- 


George McNamara, PhD
Baltimore, MD 21231
[log in to unmask]
https://www.linkedin.com/in/georgemcnamara
https://works.bepress.com/gmcnamara/75   (may need to use Microsoft Edge or Firefox, rather than Google Chrome)
http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650
http://confocal.jhu.edu

July 2017 Current Protocols article, open access:
UNIT 4.4 Microscopy and Image Analysis
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/abstract
supporting materials direct link is
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/full#hg0404-sec-0023
figures at
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/figures