[Histonet] Cryojane bone frozen sections and immunofluorescence

[Gayle Callis]

Everyone wrote: 

 

Hi Ana,

 

The OCT on the coated slides will not disappear like it does on regular
glass slides. It appears to polymerize along with the coating, but it
doesn't seem to interfere on the tissue section with the staining.

 

According to Gayle Callis' recommendation, it's best to try using the least
amount of coating rather than more. More doesn't always mean better
adhesion, and as Nancy Thomas reported usually ends up causing more imaging
interference and stain uptake making for a messier slide.

 

Teri Johnson

Manager, Clinical Trial Testing

Genoptix, Inc., a Novartis company

BioPharma

1811 Aston Avenue

Carlsbad, CA  92008

USA

 

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:
<http://lists.utsouthwestern.edu/mailman/listinfo/histonet> amaluenda at
svi.edu.au] 

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 Maluenda 

 

****************************************************************************
******************************************************

 

To Teri and all, 

 

Thanks Teri for reiterating my suggestions along with more information. 

 

We found the 4X coating to be unacceptable.  It is  more sticky but the
polymer is too thick and will cause more background when one needs to deal
with for immunofluorescence work.    With murine turbinates, unfixed and
calcified, we did use the 1X and even 1/2X but it was necessary to flash the
UV three times, waiting 30 sec between flashes so the capacitor could build
up charge.   It takes patience.   We sectioned at 5 µm but the d profile
tungsten carbide knife was very sharp so do not cut on an edge you use for
trimming into the block.  Careful removal of the pink tape is required,
inside cryostat chamber, brace the corner of slide, then pull pink tape
diagonally across the section from one corner to opposite corner.  You have
to play with temperatures with everything the same temperature.   For
turbinates -30C worked well, but tape, slides, rollers, etc. blade and
samples were at that temperature, including the UV platform.  Sectioning
temperatures vary with different laboratories, the sections can be air dried
like any other frozen section destined for solvent fixation.  I would go
ahead and air dry a frozen section and store at -80C, not -20C since the
colder temperature is more suitable for retaining antigenicity.   We
preferred using fresh, unfixed tissues, snap frozen correctly (not in a
cryostat!) over NBF or PFA prefixed/cryoprotected snap frozen bone as we
found the fresh tissue frozen section stayed transferred to the slide
better.   Other may have a different experience.   

 

You cannot remove the OCT from the polymerized surface and don't need to do
that.  There are some things your researchers will have to live with.  1)
polymer exists even with 1X, but focusing on the plane of the section and
what is in the section will work.   2) make sure you work with the brightest
fluorophors i.e. Alexa dyes.  3) autofluorescence is caused by many things
and if your sections are prefixed with NBF or PFA, then aldehyde induced
auto-fluorescence will happen but can be treated.  Go on web and get
Autofluorescence causes and cures pdf.  4) if you work with Near infra-red
fluorophores, there is no auto-fluorescence in the NIR region but the eye
can't see it but the photos are spectacular.  5) Totally  fill in those
weird polymer gaps, be overly generous with antifade mounting media. i.e.
Molecular Probes AntiFade Reagent, ready to use.  6)if working with
confocal, you can rule out backgrounds/autofluorescence or with spectral
imaging.   We learned to ignore some of the polymer background and used only
the brightest fluorophores.   Alexa dyes and Dylights are two or many
available now.   What you want is a fluorophore signal is brighter than the
goo and or autofluorescence caused by aldehyde fixation.    This goo
background problem is ongoing and has been a common complaint for years but
if you need the undecalcified bone section, you learn to live with it.
Sadly, this remains unchanged so we live with some trade offs.     We have
worked with 1/2X coating with success on murine bone but extra UV flashes
had to be done.  Turbinates are easier than tibia and femur to transfer, but
it takes practice and as said before,  patience.   

 

Normal hydration occurs but OCT is glued to slide along with section, and
never dissolves away.   If you find Cryojane to be totally unacceptable,
check out this website and how they use the Kawamoto film method that
results in some spectacular cryosectioning of bone along with beautiful
immunofluorescent staining.  Go to this site for the whole methodology
outlined in how to detail.  http://bonebase.org/histomorphometry    This
group has some excellent publications too. 

 

I hope this helps.

 

Gayle M. Callis

HTL/HT/MT(ASCP)