RE: [Histonet] vibratome or frozen sections for immuno
Geoff, if I sent 30 or 40 refereces that used both frozen cryostat sections and Vibratome sections, would you have time look them up and see what comparison was mentioned? I would also be interested in knowing about the tract tracing studies. Did they favor the Vibratome or the cryostat, and why?
Certainly, if a freeze thaw cycle is to be used anyway, initial cutting with a Vibratome makes little sense as opposed to a cryostat, unless quite thick sections are needed.
But Vibratomes are widely used for immunoreactions, and I have heard via the grapevine that they get better results. Why is that? Does everybody feel the need to permeablize? How big are the antibodies? Bigger or smaller than the target proteins? Antibodies get better access maybe, but the target protein gets a chance to leak out, and be found on a nearby cell that did not produce it. Vibratomes keep cell membranes more intact.
I agree with your comments on HRP, clearly, no need to permabilze the cells. Keep the protein in, the little molecules will find their way.
Charles W. Scouten, Ph.D.
5918 Evergreen Blvd.
St. Louis, MO 63134
Ph: 314 522 0300
FAX 314 522 0377
From: Geoff McAuliffe [mailto:firstname.lastname@example.org]
Sent: Monday, April 19, 2004 5:17 PM
To: Charles Scouten
Cc: Mike King; email@example.com
Subject: Re: [Histonet] vibratome or frozen sections for immuno
Re: the Vibratome v. frozen sections for immunostaining discussion.
I looked up the papers Charles mentions below. The first two deal with tract tracing and thus do not pertain to the original question 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 between their results with the two types of sections. Patel et al., Neurosci. Lett. 63:185-190, 1986 uses both types of sections, but their Vibratome sections are 50-100 microns while the cryostat sections are
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 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 insure that the very large antibody molecules penetrate into the cell cytoplasm. This is usually freeze-thaw (usually after appropriate cryoprotection), Triton, Tween, buffered ethanol treatment or some combination of these. However, for neuronal tract tracing large molecules need not penetrate the cell membrane, the HRP (or whatever
tracer) is already in the cytoplasm and only DAB and peroxide, relatively small molecules, need to penetrate the cell. At the same 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 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 firstname.lastname@example.org . 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.
>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 email@example.com 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).
>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
>[mailto:firstname.lastname@example.org] On Behalf Of Mike
>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) ...as 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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