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Hi Timothy,
I am a fan of Alexa Fluor 555 or Alexa Fluor 568. Depending on the
instrument (excitation source, emission wavelength selection, detector
sensitivity), and detection antibody, one may be better than the other.
Cy3 is still very popular, and similar spectra to Alexa Fluor 555.
Advantages include (i) green (Alexa Fluor 488, EGFP, mNeonGreen,
mClover, UnaG) often has overlap with autofluorescence, (ii) NIR channel
(Alexa Fluor 647, Cy5, etc ... see NIRvana Sciences for new NIR
fluorophores) is typically not visible to the eye, so hard to search.
there are filter sets that enable some users to see Cy5 by eye - see
Direct eye visualization of *Cy5* fluorescence for immunocytochemistry
and in situ hybridization. <http://www.ncbi.nlm.nih.gov/pubmed/10681398>*
Ferri* GL, Isola J, Berger P, Giro G.
J Histochem Cytochem. 2000 Mar;48(3):437-44.
PMID: 10681398
Quadruple immunofluorescence: a direct visualization method.
<http://www.ncbi.nlm.nih.gov/pubmed/9016305>
*Ferri* GL, Gaudio RM, Castello IF, Berger P, Giro G.
J Histochem Cytochem. 1997 Feb;45(2):155-8.
PMID:
9016305
I am also still a big fan of tyramide signal amplification - available
from both Invitrogen and PerkinElmer - available with several
fluorophores from each of these vendors. Generally to decrease primary
antibody from conventional immunostaining, and may need to decrease the
HRP-2ndary Ab as well.
***
The Central Dogma of DNA makes RNA makes protein implies that detecting
the mRNA should also help build confidence that the immunostaining is
correct (thee are exceptions, such as short lived RNA, transfer of RNA
and/or protein from cell to cell, etc). For mRNA detection, I recommend
Stellaris RNA FISH - see http://stellarisfish.smugmug.com/
It is compatible with immunofluorescence (I would do TSA first, if
possible). Some immunofluorescence + RNA FISH galleries are at
http://stellarisfish.smugmug.com/Immunofluorescence-RNA-FISH
See also Arjun Raj's 2013 papers on iceFISH (intron detection of nascent
transcripts ... currently at 20plex), SNP FISH (talk about the need for
stringent controls!) and Turbo FISH (15 minutes from specimen on slide
to ready for image ... though 4x more reagent).
Turbo FISH: A Method for Rapid Single Molecule RNA FISH.
<http://www.ncbi.nlm.nih.gov/pubmed/24066168>
Shaffer SM, Wu MT, *Levesque* MJ, *Raj* A.
PLoS One. 2013 Sep 16;8(9):e75120. doi: 10.1371/journal.pone.0075120.
PMID:
24066168
Visualizing SNVs to quantify allele-specific expression in single cells.
<http://www.ncbi.nlm.nih.gov/pubmed/23913259>
*Levesque* MJ, Ginart P, Wei Y, *Raj* A.
Nat Methods. 2013 Sep;10(9):865-7. doi: 10.1038/nmeth.2589. Epub 2013 Aug 4.
PMID:
23913259
Single-chromosome transcriptional profiling reveals chromosomal gene
expression regulation. <http://www.ncbi.nlm.nih.gov/pubmed/23416756>
*Levesque* MJ, *Raj* A.
Nat Methods. 2013 Mar;10(3):246-8. doi: 10.1038/nmeth.2372. Epub 2013
Feb 17. Erratum in: Nat Methods. 2013 May;10(5):445.
PMID:
23416756
See also Long Cai's multiplex detection with this type of probes (in
yeast so far):
Turning single cells into microarrays by super-resolution barcoding.
<http://www.ncbi.nlm.nih.gov/pubmed/23178478>
*Cai L*.
Brief Funct Genomics. 2013 Mar;12(2):75-80. doi: 10.1093/bfgp/els054.
Epub 2012 Nov 22. Review.
PMID:
23178478
Single-cell systems biology by super-resolution imaging and
combinatorial labeling. <http://www.ncbi.nlm.nih.gov/pubmed/22660740>
Lubeck E, Cai L.
Nat Methods. 2012 Jun 3;9(7):743-8. doi: 10.1038/nmeth.2069.
PMID:
22660740
Disclosure: Our lab/dept is hosting a wet lab workshop from Biosearch
Tech (Stellaris FISH) next week.
Somewhat different probe design ("molecular beacons") enables live cell
detection:
Mechanism of mRNA transport in the nucleus.
<http://www.ncbi.nlm.nih.gov/pubmed/16284251>
Vargas DY, Raj A, Marras SA, Kramer FR, Tyagi S.
Proc Natl Acad Sci U S A. 2005 Nov 22;102(47):17008-13. Epub 2005 Nov 11.
PMID:
16284251
This paper is cute in that they designed their molecular beacons by
looking at the rules for siRNA/miRNA and breaking all of them:
Live-cell, temporal gene expression analysis of osteogenic
differentiation in adipose-derived stem cells.
<http://www.ncbi.nlm.nih.gov/pubmed/22840182>
Desai HV, Voruganti IS, Jayasuriya C, Chen Q, Darling EM.
Tissue Eng Part A. 2013 Jan;19(1-2):40-8. doi:
10.1089/ten.TEA.2012.0127. Epub 2012 Sep 5.
PMID:
22840182
An alternative to the Biosearch oligo probes approach is to use branched
DNA - see http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3338343/
(acdbio.com) and Panomics/Affymetrix QuantiGene ViewRNA ISH
http://www.panomics.com/products/rna-in-situ-analysis/viewrna-ish-cell-assay/overview
... two companies licensed the same approach. Branched DNA ISH has been
around for a long time - Dr. Farhad Moatamed, West los Angeles VA,
showed me this circa 1995 when I was at UIC (MetaMorph customer).
Enjoy,
George
On 12/3/2013 9:47 PM, Feinstein, Timothy wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> For the many of you who have more experience in histology, do you prefer any particular label for immunostaining a challenging protein? I understand that some proteins cannot be reliably imaged in their native state using confocal. Nonetheless it seems like there must be better and worse label choices for those close calls.
>
> Years ago I might have warned people away from red and far red labels due to Low PMT semsitivity, but GaAsP detectors in those channels seem sensitive enough to moot that. Red or far red seem preferable since most tissues have relatively low autofluorescence in that range. Maybe labels in the 594-647 range are now better, or was that always true?
>
> Any input appreciated.
>
> Cheers,
>
>
> TF
>
> Timothy Feinstein, Ph.D. | Confocal Manager, Van Andel Institute
> 333 Bostwick Ave., N.E., Grand Rapids, Michigan 49503<x-apple-data-detectors://0/0>
> Phone: 616-234-5819<tel:616-234-5819> | Email: [log in to unmask]<mailto:[log in to unmask]>
>
>
--
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/26/
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