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Dear listers,
Could you kindly help me with the following.
Which clearing method/solution is best suited for paraformaldehyde-fixed brain tissue? Murrey Clear? MethylSalicylate? Others? Why?
Some of the neurons are labeled with fluorescent proteins and in some cases the signal is amplified by Alexa488 conjugate anti GFP antibody labeling.
Any hints?
Thanks & regards, jens
---
Dr. Jens Rietdorf
head of microscopy unit
Novartis Foundation
Friedrich-Miescher-Institute, wro1066.2.32
Maulbeerstr.66, CH-4058 Basel, Switzerland
phone +41(61)69-75172 mobil +41 798284737
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Rietdorf, Jens
Sent: Mittwoch, 7. März 2007 08:48
To: [log in to unmask]
Subject: Re: Micron Level Registration
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Dear Gary,
Colombelli and colleagues use a pulsed UV laser to etch the cover glass. The marks created here are about 1 mikometer thin lines and can reliably be re-loclised in EM (in EM it's the replica in the embedding which is easy to spot).
The paper is submitted, so maybe contact Julien directly, works is at the EMBL in Heidelberg, Germany.
Colombelli J, Tängemo C, Haselmann U, Antony C, Stelzer EHK, Pepperkok R, Reynaud EG. A correlative light and electron microscopy method based on laser micropatterning and etching.
Methods in Molecular Biology. Submitted.
regards, jens
---
Dr. Jens Rietdorf
head of microscopy unit
Novartis Foundation
Friedrich-Miescher-Institute, wro1066.2.32 Maulbeerstr.66, CH-4058 Basel, Switzerland phone +41(61)69-75172 mobil +41 798284737
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Gary Laevsky
Sent: Donnerstag, 1. März 2007 16:23
To: [log in to unmask]
Subject: Micron Level Registration
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Hello All,
We're taking some multimodal images of skin (SHG, 2P (auto), reflectance, and Raman), 20-60x.
The SHG, 2P, and reflectance are on one scope and the Raman is on another (pretty cool images in all modes).
We've reached a quandary on how to register these images (even get near similar fields of view, which are between 50-250 um).
A pen mark would be way too big. Too many hair follicles to narrow down to. Latex beads will probably float away (the skin needs to stay hydrated). A grid coverslip? Maybe a gene gun?
The scopes are in different buildings, so stability of the "mark" IS an issue.
Any and all input is appreciated, again.
Best,
Gary
Gary Laevsky, Ph.D.
Keck Facility Manager, CenSSIS
Northeastern University
302 Stearns
360 Huntington Ave.
Boston, MA 02115
Office(617) 373 - 2589
Lab(617) 373 - 7756
Fax(617) 373 - 7783
http://www.censsis.neu.edu
http://www.ece.neu.edu/groups/osl
http://www.keck3dfm.neu.edu
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