[Histonet] Cryojane tape-transfer and hydration of sections

[Thomas, Nancy]

Ana,
We once tried 4x coated slides thinking, like you, that it would deliver a better quality section and would stay on the slide better.  Our researchers did not like them at all because of the high level of background  staining and fluorescence.  We only use 1x now.

Nancy

-----Original Message-----
From: Ana Maluenda [mailto:amaluenda at svi.edu.au] 
Sent: Tuesday, June 09, 2015 7:20 PM
To: histonet at lists.utsouthwestern.edu
Subject: [Histonet] Cryojane tape-transfer and hydration of sections

Hello Histonetters! 

I was wondering if there is anyone out there who is currently using the CryoJane Tape-transfer system for bone sections. I have been trying to get good sections for mice tibia and femur and often find myself with problems. 

I spent a good time trying to improve the quality of sections (tried different temperatures, thicknesses and speed as well as 1x vs 4x coated slides). The sections improved (although not perfect), but I was planning to move on for a staining trial. Currently, the sections are taken at 5um, in a 4x coated slide, at -26oC. However, now I get myself with a problem when hydrating the sections. At the moment, once the sections are taken, they are kept in -20oC (for short-storage). They are then taken out and left at RT for 30 min before hydration with PBS for another 30 min. I have noticed that it seems as if the OCT around the sections doesn't completely dissolve. I have already tried PBS vs dH2O and played around with times (from 5 min to overnight) with no differences. 

Has anyone had this happening before? Can it be because of the 4x coat? Is there anything I can do to? And would this be a problem for immunofluorescence? 

Any advice would be much appreciated! 

Thanks in advance, 

Ana 


Ana Maluenda 
Research Assistant 
Stem Cell Regulation Unit 



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