[Histonet] cryosections

[Kelly D Mcqueeney]

Renee,

You should insist on an in-depth training from the vendor. Ask for a 4 
hour training from their cryostat expert, not some sales person. They 
should offer it to you. The technique is standard but varies from user 
to user. You'll have to find your way.  I am going to give you our 
standard protocol for brain IHC.

 1) Remove fresh tissue, dip in mounting medium (we use Shandon 
embedding medium, or you can skip this step) and immediately freeze in 
ice-cold isopentane (keep on dry ice for 30 minutes prior to freezing). 
For rat brain, we freeze for 20 seconds. Remove from isopentane and 
place tissue in a conical tube (some people use molds). Leave the tube 
on dry ice and immediately store at -70C.

2) Remove tissue from -70C and place in -20C freezer for 1 hour to 
equilibrate.

3) Drop embedding medium on chuck, add tissue and freeze base with 
cytocool. Cover tissue with embedding medium (we dip and freeze with 
cytocool).

4) Place chuck in cryostat and section tissue. I use probe on plus 
slides from Fisher or Superfrost from VWR.

5) Once sections are mounted on slides, dry slides completely  for 1 
hour RT and store at -80C until ready to use.

6) Remove slides from freezer and thaw at RT for 15 minutes. Place 
slides in fix and stain or perform IHC.

There are many different methods for sectioning/storing, etc. Times, 
tissue, temperature and protocols will vary from lab to lab or person to 
person.

Have fun!
Kelly

Till, Renee wrote:

>Hello.  Our research group just purchased our first cryostat. Until now,
>anytime cryosections were needed we had to send out our tissues to
>another lab. Is there a good book or other source for general
>information about cryosectioning? I am the only histotech in the group,
>but have had virtually no experience with cryosections. I know the other
>general techs that also do some histology are going to come to me with
>all their questions. We basically need to know anything and everything.
>Due to the volume of tissues, sectioning won't be performed right as the
>tissues are removed from the animal. How do you store them? Do you fix,
>and when? What type of slides are best? How do you store the slides
>after cryosectioning? Are there specific considerations to take into
>account as far as fixing, or anything else depending on the tissue or
>what stains will be performed on it? For example, I know already that
>there will be some heart and aorta sectioned for fat stains, some pig
>gi, possibly mammary gland, uterus, and mouse lung with b-gal. 
>
>Thanks in advance for any help or suggestions you can give me.
>
> 
>
>Renee'
>
> 
>
> 
>
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