Hey all,

We have been having some issues with sectioning and staining rat brain tissue.  
We are doing doing a simple nissl + myelin stain.  Here are all the details:


(1) Rats are perfused with 4% paraformaldehyde.  After perfusion, brains are 
post-fixed in PFA for at least 24 hours, but some of the brains have been 
sitting in PFA for up to 1 year.  Brains are then transferred to a 30% sucrose 
solution, and allowed to sit in sucrose for at least 72 hours (although some 
brains have been sitting in sucrose for up to 1 year).


(2) Brains are removed from the sucrose solution, and the first 10 mm of brain 
tissue is "blocked".  The whole brain is embedded in TissueTEK embedding medium 
(we also have Shandon M-1 available, but have typically used TissueTEK).  The 
brain is sectioned in a cryostat set to -21 C.  Tissue sections are 40 um 
thick.  We save every tissue section from the 10 mm block of tissue.


(3) For mounting tissue, we have tried 2 different techniques.  The first 
method is to take each tissue section and store it in a small well of 0.01 M 
PBS until we are ready to mount the tissue on slides (typically less than a 
week, often only 1 or 2 days).  The other technique is to "direct mount" tissue 
in the cryostat by lowering a slide down to the stage and "catching" the tissue 
on to the slide.  Often times, when we use this second techique, we wet the 
slide with a little bit of PBS because I have found it allows the tissue to 
"catch" easier, and as far as I can tell it helps eliminate bubbles in the 
tissue.  I will refer to these two techniques as "wet mounting" and "direct 
mounting".  "Wet mounted" slides are mounted using a dish of PBS and a 
paintbrush.


(4) Tissue sections are mounted on SuperFrost Plus slides.  No gelatin needed.


(5) After mounting, slides are typically allowed to air-dry at room temperature 
for a day or two before staining.  Occasionally staining happens on the same 
day as mounting.  Also, occasionally, staining happens up to a week after 
mounting.


(6) The staining protocol is thus:


2 min dH2O - hydrate/rinse tissue

2 min 70% EtOH - dehydrate tissue to prepare for clearing

2 min 95% EtOH - same as above

2 min 100% EtOH - same as above

5 min Citrisolv - clear the tissue (we use Citrisolv instead of Xylene)

2 min 100% EtOH - rehydrate tissue to prepare for staining

2 min 95% EtOH - same as above

2 min 70% EtOH - same as above

2 min dH2O - same as above

10 min Myelin stain (we use a Cyanine R-based myelin stain, pH is 2.0)

1.5 min differentiation step (in an ammonium hydroxide solution)

2 min dH2O

20 min Cresyl Violet stain (0.1% cresyl violet acetate solution, pH is 4.5)

2 min dH2O

5 seconds 70% EtOH (dehydrate tissue for clearing)

5 seconds 95% EtOH (same as above)

5 seconds 100% EtOH (same as above)

3 min Citrisolv (clear tissue in preparation for coverslipping)


(7) Coverslipping - We coverslip using DPX as our coverslipping medium.  We do 
not allow the slides to dry before coverslipping.  We remove them directly from 
the Citrisolv and coverslip immediately.



So, now that I have described our methods thoroughly, here is the problem: We 
are getting a lot of horizontal cracking (primarily during the sectioning 
process), and a lot of curling and sections falling off of slides (during the 
staining process).  We have tried quite a bit to alleviate the problem, but not 
much seems to work.


It seems like the tissue sections falling off during the staining process may 
have been due to the direct mounting technique - or so we think. The 
explanation we have come up with is that the embedding medium was getting 
inbetween the slide and the tissue, and then causing the tissue to fall off 
during the staining process.  Is this logical?  We have stopped direct 
mounting, and that problem seems to have stopped - hopefully.


We still have a lot of curling of tissue during staining, and cracking of 
tissue during sectioning, however.  Cracking usually happens near the 
caudal/rear portion of the 10 mm block of brain (when we approach the 
hippocampus), but rarely towards the anterior portion of the brain (motor 
cortex).  Lately we have attempted sectioning at warmer temperatures (-16 C) as 
well as tried embedding only the base of the brain instead of the whole brain.  
Results are mixed so far.  Also, nothing seems to have helped the curling issue 
during staining.


I have uploaded 6 sample brain sections at this Google Photos link: 
https://goo.gl/photos/ATQQDwq6Whs8JX8J8


I am also going to try uploading them to the Histonet Images server.  This is 
my first post to this listserv, so hopefully it works!


Thanks for any advice and comments.  We are really stumped as to how to solve 
the problem.


- David












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