Re: Fixation of Cryostat Tissues

From:Geoff McAuliffe <mcauliff@UMDNJ.EDU>

Vanessa Heim wrote:

> Hello Histonetters,
> I know this is a very basic question but I really need some advice on
> basic histology.  Keep in mind I have had absolutely no formal training
> in histology and I am teaching myself the techniques of this
> science/art.  Due to the overwhelming support of many members of this
> list, I have been able to pretty well master the art of cryostat
> sectioning.  I no longer have a problem with the tissues falling off the
> slides and I can get consistenely good sections when I section without
> wasting the whole day with the cryostat.  This brings me to my current
> dilemma.  I am having trouble finding the best fixative for the tissue
> after I have done the cryostat sectioning.  I am trying to Masons
> Trichrome Statining on Penile cross-sections and I am finding that the
> tissues are being damaged in the process.  Since I have perfomed this
> staining protocol many times in the past with paraffin-embedded tissues
> and have gotten wonderful results...i suspect that the fixation or lack
> of it is the problem that is creating such horrible looking slides.
> Thank you in advance for all the help you can give me with this
> situation.
> Vanessa Heim
> Research Assistant
> Boston Univ

Hi Vanessa:

    Tissue that has been frozen, cut and then fixed will never look as good
as tissue that has been fixed first. The cells/tissues will usually look
fuzzy or smeared, not crisp like formalin-paraffin sections. This is just
the way it is. Buffered formalin fixation should be fine for your frozen
sections, Bouin's (75 ml saturated aqueous picric acid, 5 ml glacial acetic
acid, 25 ml 37-40% formaldehyde) would probably give nicer connective tissue

Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029

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