Blue angiogram in two photon microscopy

[Heping Yuan]

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Hi, I'm wondering if anyone has any ideas for a good commercial tracer for angiograms with fluorescence emission in the blue channel using two photon microscopy. We currently use FITC-Dextran and Texas Red-Dextran for angiograms in the green and red channels but for our planned multiplexing experiment we would like the angiogram to be in the blue channel.

Thanks,
Tim

[George McNamara]

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Hi Tim,

you can continue your current colors and expensive reagents for 'vessel painting' or you can go with Sigma-aldrich (aka Millipore-Sigma)  $0.50 per mouse DiI developed by Yiwen Li, Rong Wen et al. the best way to learn is to visit Rong's lab at UMiami School of Medicine (so far not overlapping with any of the Zika zones). Their 2012 paper focused on post-sacrifice (aka no longer involving animal care committee paperwork) -- Rong told me works on live mice, mostly labeling red blood cells. I suppose another way to do it would be to label some blood cells ex vivo and then inject those (though might was well just inject 1 um or 4 um fluorescent beads of your favorite color).

you can have any color lipophilic membrane dye commercially from these two companies as long as they are green, orange, red, far-red or NIR,

http://www.sigmaaldrich.com/technical-documents/articles/biowire/cell-tracking.html

https://tools.thermofisher.com/content/sfs/manuals/CellMask_Plasma_Membrane_Stains_PI.pdf

https://tools.thermofisher.com/content/sfs/manuals/mp00282.pdf

DiA in the last comes closest to what you want (single photon ex ~450 nm, emission is ~570 nm, so strictly wavelengthing, neither green, nor red). Yiwen and Rong specifically used the not-ultra-pure inexpensive DiI from Sigma-Aldrich. See the paper for details of the glucose-saline solution (important to add the DiI "fresh").

If you really need blue, consider injecting Hoechst 33342 (or 58) or DAPI.

Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI.

Li Y, Song Y, Zhao L, Gaidosh G, Laties AM, Wen R.

Nat Protoc. 2008;3(11):1703-8. doi: 10.1038/nprot.2008.172.

PMID:

 

18846097

 

Whole-mount imaging of the mouse hindlimb vasculature using the lipophilic carbocyanine dye DiI.

Boden J, Wei J, McNamara G, Layman H, Abdulreda M, Andreopoulos F, Webster KA.

Biotechniques. 2012 Jul 1;53(1). doi: 10.2144/000113907.

PMID:

 

26307258

 

Low magnification confocal microscopy of tumor angiogenesis.

McNamara G, Yanai A, Khankaldyyan V, Laug WE, Boden J, Webster K, Li Y, Wen R.

Methods Mol Biol. 2014;1075:149-75. doi: 10.1007/978-1-60761-847-8_6. Erratum in: Methods Mol Biol. 2014;1075: E1-3.

PMID:

 

24052350

2014 book chapter mentions EX VIVO (mouse brain in a 15 or 50 mL tube) labeling with H33342 ("soak" the brain for a few minutes, such as the walk from research lab building to MPEF/confocal core building):

In collaboration with Dr. Christine Brown, Renate Starr and Professor Michael Jensen, we have imaged Hoechst 33342 dye nuclear counterstaining of hemi-sectioned mouse brains, at the Light Microscopy Core of City of Hope National Medical Center. Brain nuclei were imaged on a Zeiss LSM 510 NLO confocal/multiphoton microscope in PBS with Hoechst 33342 at 10 μg/mL for 15 min, transferred to an imaging
dish with PBS, and imaged using 750 nm, ~80 MHz, ~100 femtosecond pulses with a Zeiss 10×/0.5 NA lens. On the same microscope we have performed live cell experiments with Hoechst 33342 at 0.1 μg/mL in bicarbonate free, phenol red free, tissue culture medium. This concentration was chosen because higher concentrations of Hoechst are known to inhibit normal cell behavior [ 37 ]. When using Hoechst dyes, it is important to dilute the 10 mg/mL stock solution 100-fold in water because diluting in PBS results in formation of dye precipitates.

You can also see this book chapter for nuclear staining:

Arribas SM, Daly CJ & McGrath JC (1999b). Measurement of vascular remodeling by confocal microscopy. In Confocal Microscopy (a Volume of Methods in Enzymology), Vol. 307, pp. 246–273. Academic Press, San Diego.

and  (retina injections of DAPI):

Co-release of acetylcholine and gamma-aminobutyric acid by a retinal neuron.

O'Malley DM, Masland RH.

Proc Natl Acad Sci U S A. 1989 May;86(9):3414-8.

PMID:

 

2566171

In fact, given my experience helping Yiwen and Rong with their imaging, checking the retina (in vivo and/or ex vivo) is a great place to look to see if your labeling worked well. Tissue - ex. pancreatic islets -- anterior chamber of the (mouse) eye is also a great place to image - those stories published by Abdulreda M Caicedo A et al (UMiami).


George



On 10/28/2016 2:38 PM, Tim wrote:

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Post images on http://www.imgur.com and include the link in your posting.
*****

Hi, I'm wondering if anyone has any ideas for a good commercial tracer for angiograms with fluorescence emission in the blue channel using two photon microscopy. We currently use FITC-Dextran and Texas Red-Dextran for angiograms in the green and red channels but for our planned multiplexing experiment we would like the angiogram to be in the blue channel.

Thanks,
Tim