[Histonet] Sections of poly(caprolactone) based scaffolds washing off the slide

Richard Thibault rat1 <@t> rice.edu
Wed Apr 11 16:57:04 CDT 2012

Sorry, for clarification I meant the PCL isn't* adherent to the slide and
way too* harsh

On Wed, Apr 11, 2012 at 16:41, <rat1 <@t> rice.edu> wrote:

> Dear Histonet,
> Sorry in advance for the long email.  I would like to ask for advice
> regarding some of my sections rinsing off my slides.  I generate composite
> tissue engineered electrospun poly(caprolactone) (PCL) and cell-generated
> extracellular matrix (ECM) scaffolds. With larger amount of ECM coated
> around the PCL, I have no issues of retaining the entire section.  However,
> with minimal ECM coating, the PCL portion of the section is easily washed
> off.  The PCL portion of the scaffold is a porous electrospun random fiber
> mesh with fiber diameters of 10 microns.  The method I?ve tried is detailed
> below.
> 1. Fix the composite scaffold in 2.5% glutaraldehyde for 45 minutes.
> 2. Soak in Histoprep overnight at 4 C to ensure distribution of the
> Histoprep throughout the pores of the scaffold.
> 3. Embed the scaffold and freeze into blocks.
> 4. Let the embedded scaffold sit in the -80 C freezer overnight then
> transfer to the -20 C freezer. (this helps with the sectioning of our
> scaffolds, better integration of the scaffold with the rest of the
> Histoprep)
> 5. Cryosection into 5 micron sections and place onto superfrost plus
> slides.
> After this I run into problems.  I?ve kept the slides at -20 C and let
> them warm to room temp before staining with picrosirus red or Safranin O
> etc. as well as baking them on the slide warmer at 45 C for 1 day and up to
> a week.  I?ve also tried using superfrost excell slides, poly(L-lysine)
> coated slides, unaltered glass slides, and silanized slides (I didn?t
> expect it to work since the embedding medium is hydrophilic and rightly so,
> the entire section just beaded up destroying the section).  Each of these
> processes resulted in the PCL portion of the scaffold rinsing off within 2
> or 3 steps of the stain except for the 7 day baking step, which retained
> parts but not all of the PCL portion.
> For the staining procedure, I outline the section with a Dako pen, then
> drip the water, stains, etc. into the outline but not on the scaffold
> itself using a transfer pipet until everything is covered.  To remove the
> liquid, I use a pipettor set to 200 microliters to aspirate, again away
> from the section/scaffold, but since the PCL is adherent to the slide, it
> gets aspirated off along with the water.  Using an autostainer is way to
> harsh for these scaffolds, so I reverted to careful manual staining.
> Additionally, due to the properties of the PCL, I can?t embed using
> paraffin since the melting point of PCL is 60 C and PCL dissolves in
> xylene, chloroform, acetone, DMF, and THF.  I also can?t use gelatin coated
> slides since I need to stain for the collagen in the ECM coating.
> Does anyone have any insight to help retain the PCL portion of the
> scaffolds?  Steps I could take, different slides that may work better?  I
> do know that PCL becomes hydrophilic if treated with a high normal NaOH
> (i.e. the ester bonds in the polymer breaks down to carboxylic groups), but
> I?m not sure if that could work, since if I perform the NaOH treatment
> before sectioning only the outside of the fibers will become hydrophilic
> and if I do it after sectioning, it wouldn?t affect the bottom portion of
> the section.  This could also damage the ECM as well.  I can upload
> pictures of before and after staining if it would help. I would appreciate
> any help you could suggest.
> Thanks,
> Rich
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