[Histonet] Katushka (Far-red fluorescent protein TurboFP63) ex-vivo

[Anatoli Gleiberman]

Hi Monica,

The majority of fluorescent proteins (eGFP, RFP, Cherry, Tomato) are well preserved after 4-6h fixation with 4% formaldehyde in PBS at +4 C, following PBS washing, sucrose cryo-protection and embedding in OCT. Bright fluorescence is easily seen on cryo-sections. 

Alternatively, cardiac perfusion by 4% formaldehyde on PBS followed by 1-2h post-fixation with 4% formaldehyde, washing, cryo-protection etc. works even better for the majority of rodent organs (especially for brain).

I suspect that Katushka will be preserved as well.

Also, there are quite specific antibodies for all these fluorescent proteins including Katushka (anti-tRFP antibody from Evrogen). With these antibodies you can visualize needed protein even on FFPE sections using either HRP-conjugated secondary, or (in case of Katushka) it is possible to try IF using TexasRed conjugated secondary just to keep the signal in the same channel as Katushka itself 

Anatoli Gleiberman, PhD
Director of Histopathology
Buffalo Biolabs LLC,
73 High Street
Buffalo, NY, 14203

Phone:716-849-6810x354
e-mail:agleiberman at buffalobiolab.com



-----Original Message-----
From: Monica Aguilera via Histonet [mailto:histonet at lists.utsouthwestern.edu] 
Sent: Wednesday, March 16, 2016 4:32 AM
To: histonet at lists.utsouthwestern.edu
Subject: [Histonet] Katushka (Far-red fluorescent protein TurboFP63) ex-vivo

Hi all,

does anyone have a protocol or any recommendation for tissue fixation to preserve the Katushka far red protein expressed under a promoter gene?

Many thanks!
Mònica

--
Mònica Aguilera Pujabet, DVM, PhD
Histopathology Facility
Institute for Research in Biomedicine - IRB Barcelona Baldiri Reixac, 10
E-08028 Barcelona - Spain

Tel:  +34 934033776 <%2B34%20934020546>
monica.aguilera at irbbarcelona.org <neus.prats at irbbarcelona.org> _______________________________________________
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