Can we simply plungefreeze small pieces in LN or propane?

Can we infiltrate the unfixed tissue first with sucrose as a cryoprotectant? The air pockets will no doubt cause additional problems. Would a quick exposure to a vacuum help the sucrose or fixative penetrate?

This request is a new one for me. Any and all suggestions would be appreciated

Dr. Steven Barlow
email:sbarlow@sunstroke.sdsu.edu

Freshly prepared formaldehyde should not give you any autofluorescence or quench the probe to any great extent. I have had good luck fixing probes such as various fluo-dextrans, lectins, some membrane dyes, etc.

Lung is often a pain to cryo-section. I'd start with the easiest, a PF fixed piece of tissue, freeze it in OCT and section.

Hope it works

Thomas Moninger
moninger@emiris.iaf.uiowa.edu)

Steve,
The effect of fixation will depend upon the actual fluorophore employed. There are several which survive formaldehyde fixation. Formaldehyde autofluorescence hasn't been that big of an issue for us. Glut can be a problem in the green/yellow emission range but not so bad in longer wavelengths. If her particular needs dictate, you might try Carnoy's fluid or a methanolic version. The shrinkage is considerable, but it penetrates well and allows fairly good morphology. Hopefully, you have some tissue for trials.

Glen MacDonald
glenmac@u.washington.edu

We deal with lung tissue all the time in our lab. We inflate the lung through the bronchus/trachea using 50% OCT (Cryomatrix) compound in normal saline as a cryoprotectant. We attach a syringe filled with the OCT to the trachea/bronchus and inject the stuff down the bronchial tree. We then freeze the lung over liquid nitrogen (if you submerge the lung in liquid nitrogen, the cryoprotectant expands and the lung cracks). We store the samples in - 70 freezer and then section on a cryostat. We fix the sections in acetone for 10 minutes prior to staining. We sometimes have problems with autoflourescence but depending on the wavelength they are using it can be done away with (don't use fluorescein). If you need any other info please contact me.

Mark Elliott, PhD
MELLIOTT@prl.pulmonary.ubc.ca

Some thoughts on cryosectioning lungs.....The following procedure worked very well for me when I was doing in situ hybridization in mouse lungs:

> Fix the lungs intratracheally with 4% paraformaldehyde in PBS, using gravity feed
> Fix at 4C overnight
> Rinse in cold PBS, 3 times for 5-10 minutes each
> Immerse in 30% sucrose in cold PBS, overnight at 4C or until the lungs sink
> Drain excess liquid and freeze in OCT or Lipshaw M1 (I embedded them in histology molds complete with backing rings that fit in the cryostat, then froze them in liquid nitrogen in a stainless steel tray held over a styrofoam container of liquid nitrogen.) The Lipshaw medium was easier for me to section than the OCT.
The morphology I got with this procedure was excellent - comparable to that in paraffin-sections.

Jane A. Fagerland, Ph.D.
FAGERLAND.JANE@igate.pprd.abbott.com