X-Message-Number: 24292
From: "Basie" <>
Subject: Formaldehyde Phobia
Date: Wed, 23 Jun 2004 18:42:44 -0400

I wonder if the fear of formaldehyde is more of a psychological barrier than
a logical one. To me it seems that two perfusions. One with a low %
formaldehyde and then with a freeze preventing solution to replace the
formaldehyde will result in damage that can be repaired in the future. The
question is will this combination result in less ice damage than the freeze
preventing solution alone. Probable.



http://131.220.103.1/menzel/membrane-interact/pdf/comparison_of_cryofixation.pdf.

Actin is traditionally con-sidered as one of the most sensitive structures
to aldehydes
"
Comparison of cryofixation and aldehyde fixation forplant actin
immunocytochemistry: Aldehydes do not destroy F-actinStanislav Vitha1,2,?,
Frantisek Balu?ska3,5, Markus Braun3, Jozef Samaj4, Dieter Volkmann3& Peter
W. Barlow61Institute of Plant Molecular Biology, Academy of Sciences of
Czech Republic, ?Cesk e Bud?ejovice, Czech Republic2Department of
Biochemistry, University of Nevada, Reno, NV 89557, USA3Botanisches Institut
der Universit at Bonn, Bonn, Germany4Institute of Plant Genetics and
Biotechnology, Slovak Academy of Sciences, Nitra, Slovakia5Institute of
Botany, Slovak Academy of Sciences, Bratislava, Slovakia6IACR-Long Ashton
Research Station, Department of Agricultural Sciences, University of
Bristol, Long Ashton, Bristol, UK?Address for correspondence: Department of
Botany and Plant Pathology, 166 Plant Biology Laboratory, Michigan
StateUniversity, East Lansing, Michigan 48824, USAReceived 11 November 1999
and in revised form 7 February 2000SummaryFor walled plant cells, the
immunolocalization of actin microfilaments, also known as F-actin, has
proved to be much trickierthan that of microtubules. These difficulties are
commonly attributed to the high sensitivity of F-actin to aldehyde
fixatives.Therefore, most plant studies have been accomplished using
fluorescent phallotoxins in fresh tissues. Nevertheless, concernsregarding
the questionable ability of phallotoxins to bind the whole complement of
F-actin necessitate further optimization ofactin immunofluorescence methods.
We have compared two procedures: (1) formaldehyde fixation and (2) rapid
freezing andfreeze substitution (cryofixation), both followed by embedding
in low-melting polyester wax. Actin immunofluorescence insections of garden
cress (Lepidium sativum L.) root gave similar results with both methods. The
compatibility of aldehydeswith actin immunodetection was further confirmed
by the freeze-shattering technique that does not require embedding
afteraldehyde fixation. It appears that rather than aldehyde fixation, some
further steps in the procedures used for actin visualizationare critical for
preserving F-actin. Wax embedding, combined with formaldehyde fixation, has
proved to be also suitable forthe detection of a wide range of other
antigens.IntroductionImmunocytochemistry is an invaluable tool for studying
thein situ localization of many proteins. A plethora of additionaltechniques
are also being used to detect distributions of rel-evant molecules. For
instance, the natural dynamism of pro-teins can be visualized in vivo by
using fluorescent analoguesintroduced via microinjection (Hepler & Hush
1996) and byfusion with green fluorescent protein (GFP) (e.g., Kost et
al.1998). These newer techniques are an important complementto previously
established methods of immunocytochemistryor affinity cytochemistry in
tissues that have been subjectedto fixation and/or embedding and
sectioning.Organisation of actin microfilaments is most often stud-ied using
two methods: (1) fluorescent-labelled phal-loidin (rhodamine-phalloidin),
and (2) immunofluorescencewith anti-actin antibodies. Each method is prone
to arte-facts. Therefore, critical evaluation and comparison of themethods
are essential for the correct interpretation of theresults obtained. The
rhodamine-phalloidin technique is notwithout problems. For example, maize
root cells treatedwith cytochalasin D show nuclear accumulation of
maizeactin-depolymerizing factor (ADF), together with G-actin,which form
short actin-ADF rods. Rhodamine-phalloidindoes not bind to these actin-ADF
rods (Jiang et al. 1997).Moreover, at least in some cell types, phalloidin
does notbind to the whole complement of F-actin due to the maskingof
phalloidin binding sites with other actin-binding proteins(e.g. Nishida et
al. 1987, Ao & Lehrer 1995, Jiang et al. 1997).As noted by Staiger & Schliwa
(1987), it is disturbing that thestaining patterns obtained with fluorescent
phalloidin are ofrather a diffuse nature. Furthermore, there are often
seriousproblems with high background fluorescence as well as withrapid
fading of the signal with fluorescently tagged phallo-toxins (La Claire
1989, McCurdy & Gunning 1990). The useof actin antibodies is, therefore,
preferable.Classical immunochemical procedures using fixed andembedded
tissues provide only a static picture of proteinlocalization. The ultimate
aim of chemical fixation is toimmobilize proteins in the same state and
location as theywere in vivo. However, the fixation step is considered to
bethe most critical and there is a justified concern about theslowness of
chemical fixation which thus leaves time forabnormal re-arrangements of the
cytoplasm and cytoskeleton
----
Page 2
458S. Vitha et al.(Mersey & McCully 1978, He & Wetzstein 1995, Doris &Steer
1996). The slowness of fixation is especially criticalin the case of higher
plant cells, which are encased withinrobust cellulosic cell walls.
Therefore, alternative methodsof plant tissue preparation have been sought,
one of thembeing rapid freezing followed by freeze-substitution
(furtherreferred to as cryofixation; e.g. Baskin et al. 1995, Roy et
al.1997). However, proteins are not crosslinked by this proce-dure during
the first critical fixation step. As emphasized byMelan & Sluder (1992),
unless the fixative completely immo-bilizes soluble proteins, further sample
preparations and dif-ferential extraction could lead to artefactual
redistribution ofthe antigens. Furthermore, Wasteneys et al. (1996) noted
thatone undesirable by-product of cryofixation was a punctate-like
appearance of microtubules.In chemical fixation, there is the serious danger
that aldehy-des may damage the antigenicity of some epitopes and henceimpair
their binding of antibodies. Actin is traditionally con-sidered as one of
the most sensitive structures to aldehydes(Lehrer 1981) and the early
difficulties of visualizing F-actinin plant cells

Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=24292