Quantum dot (Qdot) penetration in brain slices and fluorophore recommendation

[Matiar Jafari]

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Dear ListServ,

We recently purchased some Qdots (Life Technologies) to do some secondary
antibody labeling as their Qdot 625 fell really nicely into our emission
filters range  (624 filter / 40 bandpass), however we only get at best 12
um of penetration cumulatively (6 um front of slice and 6 um back of
slice).  I was wondering if anyone here could comment on the penetration
and if they have any comments on how to get better results.  We've even
tried longer incubation, greater concentration of permeabilization reagent,
and longer permeabilization reagent incubation times all to no avail.

Secondly, I was wondering if anyone could recommend a secondary fluorophore
that has a relatively high two photon action cross section, with good
penetration (at least through 25 um of brain tissue), and that fits well
into our emission filter's range (624/40).

Thank You
-------
Matiar Jafari

[Ray Gilbert]

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Hi Matiar

I tried Q Dots on formalin fixed, human brain slices a few years ago without a lot of success. We tried both conjugated secondaries and strep linked dots but like you found penetration an issue. We also got high background sometimes although there did not seem to be any pattern to when this appeared or didn't. We got some hot spots where penetration seemed much higher and I suspect they may have been areas where the tissue was damaged or torn allowing them more room to enter

We had some success on paraffin embedded sections. In the end we gave up on trying and stuck with the tried and true Alexa and Cy dyes we were working with.

Thanks

Ray



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Matiar Jafari
Sent: Wednesday, 6 June 2012 12:04 p.m.
To: [hidden email]
Subject: Quantum dot (Qdot) penetration in brain slices and fluorophore recommendation

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear ListServ,

We recently purchased some Qdots (Life Technologies) to do some secondary
antibody labeling as their Qdot 625 fell really nicely into our emission
filters range  (624 filter / 40 bandpass), however we only get at best 12
um of penetration cumulatively (6 um front of slice and 6 um back of
slice).  I was wondering if anyone here could comment on the penetration
and if they have any comments on how to get better results.  We've even
tried longer incubation, greater concentration of permeabilization reagent,
and longer permeabilization reagent incubation times all to no avail.

Secondly, I was wondering if anyone could recommend a secondary fluorophore
that has a relatively high two photon action cross section, with good
penetration (at least through 25 um of brain tissue), and that fits well
into our emission filter's range (624/40).

Thank You
-------
Matiar Jafari

[haisond]

I have had success at Qdot labeling in brain slices as thick as 80 microns.  I use a biotinylated anti-NeuN primary ab and Qdot 525 secondary.  It will require 1 week of incubation with the primary ab and an additional 1 week with the Qdot secondary.  Use 1% TritonX 100 in TBS with 2% normal goat serum as the primary ab diluent and TBS with 5% BSA (IgG free, Jackson Immunolab) and 0.1% TritonX 100 for the Qdot diluent. Also, rotating your samples during incubation is a must, I use a 360 degree rotator to ensure that my samples are agitated uniformlly.  Following primary ab incubation, a 24 hr wash (TBS 0,1% TritonX100 with sodium azide (0.05%) and thymol (100 ug/ml) is required to reduce high background (the azide and thymol is for inhibition of mycoplasma and bacterial growth). For long incubation, I also add sodium azide (0.05%) to all the reagents to prevent bacteria from growing.  With this protocol, I've had success with multilabeled Qdots in 40 micron sections.  Good luck.