I have done immunostaining on free floating scetions of Rat brain.
Tissue fixed, by perfusion in 4% Para, then cryo protected with 30%
sucrose. Sections cut at 50um, then placed into 500ul PBS in a 24-well
plate. (I had approx 4-5 sections per well)

500ul is plenty in a well, especially if you are using a fair dilution
of antibody cos then you dont use too much.

I did all my staining in the plate, and the sections looked wonderful!

Hope this is of some help,

emma carter

> -----Original Message-----
> From:	Cynthia Favara [SMTP:cfavara@niaid.nih.gov]
> Sent:	Tuesday, January 25, 2000 9:16 PM
> To:	'HistoNet Server'
> Subject:	free floating sections
>
> All,
> 	I have been asked to stain free floating sections and am
> enlisting
> my greatest resource. If possible we would like to use frozen tissue
> and
> thick sections. The premise is that we can stain without the tissue
> ever
> drying. Advice from anyone with experience would be greatly
> appreciated.
> I'll take any ideas and promise not to be offended by sarcasm etc
> [polly
> would definitely freeze here today]
> TIA,
>
> Cynthia Favara
> Rocky Mountain Laboratories
> 903 S 4th Street
> Hamilton, MT 59840
> ph: 406-363-9317
> FAX: 406-363-9286
> e-mail: cfavara@nih.gov