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

rat1 <@t> rice.edu rat1 <@t> rice.edu
Wed Apr 11 16:41:07 CDT 2012

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  
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.



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