Re: [Histonet] vibratome or frozen sections for immuno

From:Geoff McAuliffe

Re: the Vibratome v. frozen sections for immunostaining discussion.

    I looked up the papers Charles mentions below. The first two deal=20
with tract tracing and thus do not pertain to the original question=20
about penetration of antibodies/immunostaining reagents into 50 micron 
sections. The second two papers use both Vibratome and frozen sections. 
Hartig et al.,  J. Neurosci Meth. 67:89-95, 1996 finds no difference=20
between their results with the two types of sections. Patel et al.,=20
Neurosci. Lett. 63:185-190, 1986 uses both types of sections, but their 
Vibratome sections are 50-100 microns while the cryostat sections are=20
2-6 microns. This does not seem like a valid comparison to me. However, 
they did not mention any difference in results between one type of=20
section and the other.
    I think confusions arises in the intended purpose of the sections. 
Certainly for immuno some sort of permeabilization is usually used to=20
insure that the very large antibody molecules penetrate into the cell=20
cytoplasm. This is usually freeze-thaw (usually after appropriate=20
cryoprotection), Triton, Tween,  buffered ethanol treatment or some=20
combination of these. However, for neuronal tract tracing large 
molecules need not penetrate the cell membrane, the HRP (or whatever=20
tracer) is already in the cytoplasm and only DAB and peroxide, 
relatively small molecules, need to penetrate the cell. At the same=20
time, one does not want the tracer to leak out of the cytoplasm. Perhaps 
Vibratome sections are more appropriate for that task but I suspect=20
other variables would need to be considered as well


Charles Scouten wrote:

> Numerous studies have made the comparison. See a selected few below.  
>It has been popular wisdom for 20 years that Vibratome sections, by not facturing the cell membrane, preserve the cytosol better and give more intense staining of cytosol proteins.  I have personally seen differences with HRP staining, but didn't try to prove that some other variable was not responsible.
>I have located the studies below, and many others, that have made the comparison.  I do not have access to the complete text, and do not know if all of these say the vibratome results were better.  I would not expect vibratome to do better with membrane bound proteins.  
>The science is there and done, I would compile a complete list of recent references like those below (maybe about 20-30) if somebody will volunteer to look them all up in Science Direct or the original Journals and check whether Vibratome sectioning was better, worse or the same compared to cryostat sectioning.
>Current concepts in neuroanatomical tracing
>C. Köbbert, R. Apps, I. Bechmann, J.L. Lanciego, J. Mey & S. Thanos Progress in Neurobiology, 2000, 62:4:327-351
>. The success of labelling can be estimated roughly, and the tissue can be sectioned with a vibratome. Cryosectioning of the tissue is not recommended, since some dye leakage occurs during the procedure.=20
>Neural tract tracing using Di-I: a review and a new method to make fast Di-I faster in human brain
>D. Larry Sparks, Lih-Fen Lue, Timothy A. Martin & Joseph Rogers Journal of Neuroscience Methods, 2000, 103:1:3 - 10
>if a fluorescent Di-I product is desired, infiltrating and embedding brain tissues in polyacrylamide prior to sectioning by vibratome eliminates the necessity of dehydrating and clearing the tissue sections prior to coverslipping ( Hayaran and Bijlani, 1992).
>(too thick)
>Although the thickness of the vibratome sections (50 µm) was less than optimal for  demonstrating individual axons, individual fiber tracts could occasionally be traced for as much as 5 mm or more, as shown here. Cryostat and other techniques were used to achieve thinner sections, but requiring freezing of the tissue, resulted in diffuse smearing of the fluorescence label
>Somebody let me know if this is favorable to my case.  The comparison was made, but I can't get the full text to see how it came out.
> Triple immunofluorescence labelling of parvalbumin, calbindin-D28k and calretinin in rat and monkey brain 
>Hartig, W. / Bruckner, G. / Brauer, K. / Seeger, G. / Bigl, V., Journal of Neuroscience Methods, Aug 1996
>...Thereafter, two brains were cut on a Vibratome (TSE, Kronberg) at 50 1lm thickness...monkey brains the tissue was cryo-protected by equili- bration...about 4 days and subsequent cryo-protection. 2.3. Tnple immu7...results were obtained with Vibratome and frozen sections and with... 
>  53.  Adenosine deaminase and histidine decarboxylase coexist in certain neurons of the rat brain 
>Patel, B.T. / Tudball, N. / Wada, H. / Watanabe, T., Neuroscience Letters, Jan 1986
>...further 24 h at 4?C. Thick vibratome sections (50-100/~m) were obtained...required, the fixed blocks were cryo- protected with 301~:~ sucrose...and dried at 20~C for 24 h. Vibratome and cryostat sections were...immunoreactivity in rat brain vibratome sections was consistent with... 
>Charles W.  Scouten, Ph.D. 
>5918 Evergreen Blvd. 
>St. Louis, MO 63134 
>Ph: 314 522 0300  
>FAX  314 522 0377 
>-----Original Message-----
>From: [] On Behalf Of Mike King
>Sent: Wednesday, April 14, 2004 12:56 PM
>Subject: [Histonet] vibratome/frozen immuno, soap residue, autofluorescence, and bad list posting habits
>re 9. RE: Vibratome Considerations (Charles  Scouten) for immunolabeling sensitivity differences between vibratome and frozen sections from cryoprotected tissue, is there any science to support this claim (references)?  i've never seen a difference and doubt there is really any reason to expect one.  especially if you need to permeabilize anyway...
>re Danielle Zalinski Vibratome Considerations post:
>...50 micron sections of brain (vibratome or other) will label through the entire thickness depending on the fixation, permeabilization, and location of the antigen.  if you're going to use the tissue for anything that demands full-thickness labeling, you should use a confocal microscope to check that the labeling is even through the entire thickness of the sections.  papers in the histo literature will detail the procedures you need for your material, or post again for specifics.
< other stuff deleted in the interest of brevity.

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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