Re: [Histonet] SEM for collagen fibers

From:"Hernan Aldana Marcos"

You have a lot of methods
I send two:
1- Tissues was fixed by the immersion procedure (gluta or parafor). It was
dehydrated in a graded series of ethanol, and fractured with frozen ethanol.
The samples were dried by the critical point method with carbon dioxide.
Samples were mounted on aluminum stubs, sputtercoated  with gold, and
examined in a Jeol JSM-15 scanning electron microscope.

2- Tissues were transferred to phosphate buffer (pH 7.4) for 2-3 d to remove
excess of glutaraldehyde and soften, before dehydrating throught alcohols,
clearing in amyl acetate and embedding. Tissues were sectioned at 5 µm until
some particualr feature (such as the gland duct) was reached. The final
section was then stained with H-E to act as a reference and unsectioned
tissue reprocessed for SEM. Wax was removed by trimming, by heating at 56°C
and finally by tranferring to xylene at 37°C, receiving 4-5 changes of
xylene over 24-36 h. Tissues were placed in 2 washes of absolute alcohol,
critical point dired, mounted and coated as a usuall. (J. McGadey et al. J
Anat (1992) 180. 127-136.)
3- we do the two techniques with excelllent results. Also we make a
modification in the technique two. When I make the bloks in parafin I
fracture the block with my hands or using the "floor shock" and then make
the reprocessing to SEM.
Dr. Hernán J. Aldana Marcos
Facultad de Medicina. Universidad de Morón
Machado 914. B1708JPD. Buenos Aires. Argentina
e-mail alternativo

----- Original Message -----
Sent: Wednesday, December 10, 2003 9:05 AM
Subject: [Histonet] SEM for collagen fibers

> hello dear histonetters
> first many thanks for your last precious help
> I have few questions about identification of collagen fibers in
resconstructed tissue
> Do you think that fixation of small tissue with glutaraldéhyde for three
days can avoid proper stainning of collagen fibers with trichrome stains,
should i change the fixative to paraformaldéhyde for instance or reduce time
of fixation ?
> Do you know if it's possible to do SEM observations on sections which have
been previously embedded in methylmetacrylate or paraffine ?
> the issue is as well to observe collagen fibers and their orientation
> i have no experience to doing that, does someone have a protocol ?
> thank you so much for anyhelp
> Myriam baali
> Natural implant
> 163, av de luminy
> marseille, france
> tel: 04 91 80 41 89
> _______________________________________________
> Histonet mailing list

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