Dear Rose, 

congratulations for your ability to cut 70µm thick sections of human brain....

Before one can comment seriously on the problem you face and get an idea about how to get rid of those problems there are left some questions:       As you wrote

"After staining I mount section on coated slide and the counterstain is taken up by the surrounding gelatin matrix and it does look unattractive."  -->   Assuming you stain your brain section(s) therefore FLOATING on a hydrous staining solution - then taking those section(s) with further washing(s) unto your coated slide...  "coated"  with what?...

Further/secondly "This gelatin seems to be thicker than 10% and I do know the specimen was post fixed in block before frozen sectioning making the gel tougher. " : you should get MORE information on that treatment.... I know there are several methods for "fortification/strengthening" of the tissue blocks using gelatine soaking and afterwards crosslinking the gel(atine) somehow either by fixation (formaldehyde, or also glutaraldehyde), or by treating with other crosslinking agents. So you think, "this gelatin" (gel, of the block, afterwards=the achieved section?) "is thicker than 10%" (and perhaps should be ?? %?). 

It remains a bit unclear [except for the fact(s) that you HAVE folds in the sections]: How could folds form in a gel(atine) matrix during optimal mounting ex water/staining bath?)? 

And why you don't counterstain [which one] your floating sections after the staining with what?...) how the counterstain is taken up into the [I guess - brain section proper-] surrounding gelatin matrix  and - due to the uncertain fact that you cut cross-linked gelatine - and use cryosectioning "making the gel(atine?) "tougher" -  it might be that the crosslinking isn't reversible (to be assessed and proven) ... 

IMHO, it would be the FIRST action that your client(who prepared specimens for you to section the blocks) reveals "in toto" and comprehensible the method used to arrive at those blocks of brain you section at 70µm thickness [BTW, last question: how big are they in their size/dimension?: mm or cm? Thank you).

Hello, Rose! I know what you mean. I got some specimens of human hippocampus embedded in gelatin and had the same problem! Gelatin "auras" counterstained and folded over the tissue. They were meant to be sectioned using vibratome, but I ended up using only cryostate. I rather use tissue-tek oct in this case. Thus I started to carefully remove the gelatin block the best I could before cryosectioning or remove it while mounting sections on the slide (dipping slides on PBS on a Petri dish and gently removing the excess of gelatin from the floating section with a soft brush while mounting). It took me much more time to finish each slide but improved quality. Since you have thick slices that might not be a problem (I had 60µm thick sections). Good luck!

Thanks Luciana Pimentel-Silva!  That is in essence what I have been doing with the sections that I have been sent pre-cut.   

Wolfgang H. Muss

1.  Coated slides are done with aminosilane on pre cleaned glass.  I can get the brain tissue itself nicely spread on the slide though it does take time.  The problem does lie in the gelatin surrounding the tissue.  

2.  Unfortunately the publication only mentions formalin postfixation of the gelatin.  I am trying to contact the gentleman who did the work, but there was some controversy involving the project and it is hard to communicate directly.  One contact outside his laboratory mentioned that the gelatin might have been made at 12-15% which is believable since this was a whole human brain that was sectioned. 

3.   I stain the sections for IHC as floating sections which works very well then mount on slides.  Then a light hematoxylin counterstain is applied.  The folds are of the gelatin at the microscopic level, sometimes flipping over the edge of the gray matter where tau or senile plaques are seen.  For an H&E I pre mount the section on the aminosilane coated slide then air dry overnight.  Stain as usual for an H&E but using far larger quantity of solution. Less problems there of course but still the halo of gel around the section is unsightly even after I carefully wipe before coverslipping.

4.  Sections are big, coronal sections of human brain.  I have experience with paraffin sections of this nature so this is new for me using frozen floating sections of THIS size.  (aprox. 140mm by 120mm)

Dear Rose,

Are you using enzymatic (DAB, etc.) or fluorescence as your IHC label?  Since you are using the hematoxylin after the IHC, presumably to mark the nucleus, have you tried using a fluorescent nuclear marker (DAPI or Hoechst) to mark the nucleus?  That way, you might be able to use one of the aqueous mountants for fluorescence that might be more compatible with gelatin. 

Good Luck!

Jill

Jill, I am using DAB.  I did a thioflavin which has a gelatin mounting media and the darn matrix still showed up due to picking up a little more of the stain that could not be washed out in the alcohols and water rinses. 

I have tried warm water to rinse the section in as some have suggested on the internet, but this gelatin is very tough perhaps due to it being post-fixed in formalin and being over 10%.   Trying to get in contact with the gentleman who did the sectioning, but no answer as yet.  

Dear Rose, thank you for clarifying the technique you use/used I am thinking over....but please allow some time for responding (we celebrate a holiday - All Saints' Day) today...

Best wishes,

Wolfgang