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