RE: [Histonet] vibratome/frozen immuno, soap residue, autofluorescence, and bad list posting habits

From:"Charles Scouten"

 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. 

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.

re 3. autoflourecence in RBC (James Watson) ...try 0.1% sodium borohydride for quenching autofluorescence; this used to be on the Molecular Probes website.  please post your results if this does/doesn't work.

re 13. Clean Glassware Test (Ostrander, Anita B) ...for testing for soap residue, any bartender worth his/her salt will tell you that nothing takes the head off a fine beer like a trace of soap on a poorly rinsed glass. perhaps your lab should stock a few pints of Guinness?

...and please, please, please, kind histonetters, don't include the whole digest in replies to histonet.  hardly a day goes by when the digest doesn't contain at least one complete copy of all the previous day's posts because somebody didn't think before replying.  we've all hit the send button too soon, but this is a real nuisance.

happy histo,

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