Re: Nissl stain

From:Donna Simmons <>

Hi Joyce,
I'll send the fax today.  Other HistoNetters with more experience 
using Paraffin Embedding for brain [NeuroPath types?] can probably 
give you some hints on eliminating wrinkles.  Sounds as if you may 
have the infamous "poor fixation/infiltration in the center of the 
block" problem...  Are you optimal for block size, fix and processing 
times, and knife sharpness?  What species?  Primate is much like 
human, I think -- but rodent requires a different processing schedule.

If your fixation and processing are good, here's a trick I have used:
Lay a section horizontal on a large slide, then gently float it in 
20% alcohol [I use ethanol] by using a squirt bottle to direct a flow 
of alc under it.  (I keep a 2x3in slide sitting atop a 50 ml beaker 
for drainage, and a squeeze bottle of 20% alcohol beside my 
waterbath).   Tilt the slide to gently drain away the alcohol.  Then 
immediately float the section on the waterbath by lowering it SLOWLY 
at an angle.  Careful! --the difference in surface tension between 
the alc and water will create turbulence that flattens the tissue, 
and can make the section go spinning across the waterbath if you 
don't control it with a brush or other 'stopper'.  You'll need to 
practice, and make sure your water bath is at the correct temp -- 
cooler for brain than for other paraffin embedded tissue.  You may 
need to adjust alc concentration [down] to get the perfect balance of 
turbulence and heat-flattening.  You're adding alc to the waterbath 
each time you float out a section, so over the day you may need to 
change the water or up the concentration of alc.  This technique is a 
bit slow, but it has been a lifesaver when serial sections are 
absolutely essential, or when you just cannot get one perfectly flat 
paraffin section.   I've even used it on ribbons of smaller size 
tissues like pituitary or embryonic brain.

PS:  just read the HistoNet Daily Digest from yesterday, and I wonder 
if anyone knows what was the strange MIME attachment to my message? 
This was the first time I tried emailing to the HistoNet from home by 
modem, but I used my regular Eudora email, not a browser that uses 

PPS: Lori Gibbs,   Hi! -- glad to see you responded to the Nissl 
query, too.  Fun to see again the tried and true cresyl method for 
monkey brain that I used years ago at the UW Primate Center...  ;-)

>Simmons and Swanson, "The Nissl Stain" in
>Neuroscience Protocols, 93-050-12-01-07, Elsevier press, 1993.
>I would appreciate the information also.  If you would fax a copy also.  Name
>is Joyce Christopher Fax number is 913-433-5125.  Thank you for sharing.
>Would you also have a suggestion for sectioning brain without wrinkles.  We
>perfuse with 4% paraformaldehyde, process on VIP, formalin, 70-100, xylene and
>paraplast extra.  Timing is 40 minutes per station, ambient and no 
>pressure.  I
>have a terrible time with wrinkles especially sections from the center of the

<< Previous Message | Next Message >>