[Histonet] 1% gelatin/40% etOH mounting solution

[Maria Mejia]

Brandon,

Taught yourself some histology techniques - good for your!!! I use 
gelatin sub
slide that I do in-house. It works for me. I start with clean slides and 
then
I wash them...again...myself with a soft brush & liquid-Nox. I wash well 
with
plenty of tap H20 (running water - 30mins to 1hr.) Then rinse well with 
DH20.

While cleaned slide are setting in DH20, I make the gelatin solution 
using Sigma
300 bloom (since your using brain sections at 40 ums thick).
 > cold distilled water - 500ml
 > 300 bloom gelatin 5gm

Mix well w/stirring bar & heat - make sure that solution is not over 
heated above
60C (cause it will denature the protein in the gelatin). I keep the temp 
at about 58C.
**If gelatin solution starts to get cold, just put back into oven to 
keep warm and
use when ready.
 > add 0.2 gm of chromium potassium sulfate. Mix well.
 > add a few grains of thymol for preservation.
Filter solution using #4 filter paper and store in oven at about the 
same temp - 58C.

Can do single or double coat of gelatin on slides:
 > dip slides either using slide rack or can also do by hand each slide 
individually
dip each slide 10 times and dry slides up-right.
 > once slides are completely dry store in clean slide boxes and date. I 
keep gelatin
dip slides only for 6 months.
**If still having problems with sections falling off, repeat the 
procedure twice, so there
is a second coat of gelatin on each slide with drying time in-between.

But, I'm sure this will work for you! If you get any staining 
background, I'd give
Gayle's  method of reducing background a try! I sure will.

Hope's this helps.

Maria bartola Mejia
Smith-Kettlewell Eye Research Institute
San Francisco, CA 94115
Email: maria <@t> ski.org
Phone: (415)-345-2185


Brandon Munk wrote:

>I am a behavioral ecologist learning histology for a research project
>sectioning bird brains and staining with cresyl violet. Birds were
>transcardially perfused with 4% paraformaldehyde in the field, brains
>removed and post-fixed for about a week before being stored in PB until
>sectioning. Cryoprotected in 20% sucrose/PB solution until brains sink,
>embedding in OCT and sectioned on freezing stage microtome at 40 um taking
>every fifth section. Mounted on plus slides and stained with cresyl violet
>for volumetric measurements of brain regions. I spent much of my summer
>practicing sectioning and staining on house sparrows to fine tune my
>protocol which got me to the microtome (over cryostat) and non-subbed plus
>slides seemingly working best (now obviously not the case!).
>
>Brandon
>
>P.S. I tried your cresyl violet staining protocol and it worked wonderfully,
>thanks!
>
>
>  
>
>>From: Maria Mejia <maria <@t> ski.org>
>>Date: Tue, 27 Sep 2005 10:51:34 -0700
>>To: Brandon Munk <bmunk <@t> uwyo.edu>
>>Cc: <histonet <@t> lists.utsouthwestern.edu>
>>Subject: Re: [Histonet] 1% gelatin/40% etOH mounting solution
>>
>>Brandon,
>>
>>I have a few questions for you. Please, tell me what was the tissue
>>fixed in?
>>What's the tissue that your working with? How thick are these sections? Are
>>these sections FFPE (formalin-fixed paraffin embedded)? And, finally what
>>do you plan to do with the sections, i.e. IHC or other?
>>
>>Regards
>>
>>Maria Bartola Mejia
>>Smith-Kettlewell Eye Research Institute
>>San Francisco, CA, 94115
>>Email: maria <@t> ski.org
>>Phone: (415)-345-2185
>>
>>
>>
>>Brandon Munk wrote:
>>
>>    
>>
>>>Histonetters,
>>>
>>>I am trying to get fixed tissue sections to stick onto slides better. I have
>>>tried subbed slides, plus slides (subbed and un-subbed) with only mediocre
>>>results. I was recently given a recipe for a 1% gelatin in 40% etOH mounting
>>>solution. This seems to work wonderfully on un-subbed plus slides, but I am
>>>having much difficulty in keeping the gelatin in solution long enough for it
>>>to be useful for mounting my sections. The recipe consists of heating
>>>distilled water (not over 60C) dissolving gelatin (enough for a 1% final
>>>concentration) and adding 80% etOH in two steps to the cooling gelatin
>>>solution, half soon after gelatin goes into solution and the other half
>>>sometime into the cooling of the solution. The recipe is vague since there
>>>is probably some voodoo that goes along with it. I have tried about 10
>>>different variations on this recipe, none work consistently. Does anyone
>>>have some tips, pointers or advice to help me out, or even another recipe
>>>that may work better. Thanks in advance!
>>>
>>>Brandon Munk
>>>Dept. Zoology and Physiology
>>>University of Wyoming
>>>
>>>
>>>_______________________________________________
>>>Histonet mailing list
>>>Histonet <@t> lists.utsouthwestern.edu
>>>http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>>> 
>>>
>>>      
>>>
>>    
>>
>
>  
>