An improved method for clearing and staining free-hand sections and whole-mount samples

BACKGROUND AND AIMS: Free-hand sectioning of living plant tissues allows fast microscopic observation of internal structures. The aim of this study was to improve the quality of preparations from roots with suberized cell walls. A whole-mount procedure that enables visualization of exo- and endodermal cells along the root axis was also established. METHODS: Free-hand sections were cleared with lactic acid saturated with chloral hydrate, and observed with or without post-staining in toluidine blue O or aniline blue. Both white light and UV light were used for observation. Lactic acid was also used as a solvent for berberine, and fluorol yellow for clearing and staining the samples used for suberin observation. This procedure was also applied to whole-mount roots with suberized celllayers. KEY RESULTS: Clearing of sections results in good image quality to observe the tissue structure and cell walls compared with non-cleared sections. The use of lactic acid as a solvent for fluorol yellow proved superior to previously used solvents such as polyethylene glycol-glycerol. Clearing and fluorescence staining of thin roots such as those of Arabidopsis thaliana were successful for suberin visualization in endodermal cells within whole-mount roots. For thicker roots, such as those of maize, sorghum or tea, this procedure could be used for visualizing the exodermis in a longitudinal view. Clearing and staining of peeled maize root segments enabled observation of endodermal cell walls. CONCLUSIONS: The clearing procedure using lactic acid improves the quality of images from free-hand sections and clearings. This method enhances the study of plant root anatomy, in particular the histological development and changes of cell walls, when used in combination with fluorescence microscopy.     

用共聚焦扫描显微镜检术观察水稻胚囊发育

介绍了一种快速简便观察分析水稻胚囊发育程序的方法。水稻子房经固定和脱水,用冬青油整体透明及丁香油封片,在共聚焦扫描显微镜检术下,发育中的胚囊产生自发荧光、可分辨出胚囊内部结构。ＦＡＡ和４％戊二醛两种固定液效果不同,后者效果较佳。     

Offsets for land clearing: No net loss or the tail wagging the dog?

Summary   Offsets (also known as mitigation banks, compensatory habitat, set-asides) is a policy instrument recently introduced in several States in Australia to permit some land clearing while striving for no net loss in the extent and condition of native vegetation overall. Offsetting is criticized with respect to the amount of gain required to compensate for losses from clearing, the equivalence of losses and gains, the time lag between losses and gains and a poor record of compliance. Despite these criticisms, we conclude that offsetting is a useful policy instrument while governments continue to permit some clearing of native vegetation. However, offsets will only contribute to no net loss if (i) clearing is restricted to vegetation that is simplified enough so that its functions can be restored elsewhere with confidence or clearing is restricted to vegetation that is unlikely to persist and is not practicable to restore irrespective of clearing; (ii) any temporary loss in habitat between clearing and the maturation of an offset, or differences between the habitat lost from clearing and gained through an offset, does not represent significant risk to a species, population or ecosystem process; (iii) there will be gains of sufficient magnitude on the offset site to compensate for losses from clearing; (iv) best practice adaptive management is applied to offsets; (v) offsets are in place for at least the same duration as the impacts from clearing; and (vi) there is adequate compliance. Land clearing with offsets outside these parameters is inconsistent with 'no net loss'.     

Techniques for Clearing Ovules for Studies of Megagametophyte and Early Postfertilization Development in Rhododendron

Using ovule clearing, more than 33,600 ovules of Rhododendron nuttallii T. W. Booth (Ericaceae) were examined for megagametophyte and early postfertilization stages at daily intervals from anthesis until 3 weeks after pollination. Pretreatments with amyloglucosidase to digest integumentary and gametophyte starch and Stockwell's bleach to remove tannins from the integumentary epidermis were necessary. Ovules were cleared by a combination or modifications of Heir's four-and-a-half or Stelly's hem-alum-methyl salicylate techniques and were observed using differential interference contrast optics. The method proved suitable for large-scale quantitative studies of ovule development and fertilization. A general protocol is suggested as a starting point for ovule clearing studies.     

Using wintergreen oil for mounting mosquito larvae: a safer alternative to xylene

Permanent mounting of fourth instar mosquito larvae is essential for identifying Aedes spp. This procedure requires extensive exposure to xylene, a clearing agent in the mounting process. We investigated wintergreen oil as a substitute for xylene. Five hundred larvae were mounted on slides to evaluate shrinkage or expansion of specimens after clearing using xylene or wintergreen oil. We examined the ventral brush and siphonal hair tufts for species identification and for preservation of morphological characteristics after clearing specimens in xylene or wintergreen oil. Shrinkage of the length of whole larvae and width of the head, thorax and abdomen after mounting was significantly greater after clearing with xylene than with wintergreen oil. The length of the comb scale nearest the ventral brush was similar for both clearing agents. The clarity of the specimens after mounting was improved by clearing with wintergreen oil, but the integrity of the ventral brush and siphonal hair tufts were similar for both clearing agents.     

Whole‐body clearing of beetles by successive treatment with hydrogen peroxide and CUBIC reagents

Internal tissues of multicellular organisms cannot directly be seen because they contain pigments. For this reason, whole‐body clearing methods have been developed and applied to mammals such as mice. Insects such as beetles, however, cannot be cleared by the mammalian method because of pigments such as melanin in their exoskeletons. In this study, we tried to develop a whole‐body clearing method for large beetles. We first bleached the exoskeleton using a hydrogen peroxide treatment, and applied advanced Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis (CUBIC) reagents to make the internal tissues transparent. The combined method of hydrogen peroxide and advanced CUBIC allowed us to successfully undertake whole‐body clearing of large beetles.     

Assessing land clearing potential in the Canadian agriculture–forestry interface with a multi-attribute frontier approach

The pattern of forest land clearing in a region can be viewed as a gauge of sustainable (or unsustainable) use of agricultural and forest resources. In this study we examine the geographical distribution of land clearing potential in the Canadian agriculture–forestry interface and propose a new landscape-scale indicator that quantifies this potential. We consider the possibility that forested land will be cleared for agriculture as a trade-off between the land's capacity to support agriculture and its comparative value if it remains forested. However, this trade-off is complicated by the land's susceptibility to fragmentation (and subsequent conversion), which derives from the local pattern of forest, agriculture and other land cover types. We find the locations in the agriculture–forestry interface with the highest land clearing potential by delineating nested multi-attribute frontiers in the dimensions of the land's agricultural capacity, its estimated forest productivity and its fragmentation potential. The multi-attribute frontier concept addresses our lack of knowledge about the relative importance of these multiple drivers of land conversion by objectively combining them into a single-dimensional land clearing pressure metric in a geographical setting. Overall, our approach provides a simple yet informative indicator which reveals the geographical stratification of land clearing pressures across large regions. In general, the spatial delineation of areas with high land clearing potential agrees well with recent evidence of land clearing and deforestation events in Canada.     

A simple and rapid microwave-assisted hematoxylin and eosin staining method using 1,1,1 trichloroethane as a dewaxing and a clearing agent

The use and practicability of microwave-assisted staining procedures in routine histopathology has been well established for more than 17 years. In the study reported here, we aimed to examine an alternative approach that would shorten the duration of dewaxing and clearing steps of hematoxylin and eosin (H & E) staining of paraffin sections by using a microwave oven. Although xylene is one of the most popular dewaxing and clearing agents, its flammability restricts its use in a microwave oven; thus we preferred 1,1,1 trichloroethane, which is not flammable, as the dewaxing and clearing agent in the present study. In Group I and Group II (control groups), intestine was processed with xylene and 1,1,1 trichloroethane, respectively. The sections were then stained with H & E according to the conventional staining protocol at room temperature and subdivided into two groups according to the duration of dewaxing and clearing in xylene. In Groups III and IV (experimental groups) similar tissues were processed with xylene and 1,1,1 trichloroethane, respectively; however, sections from these groups were divided into four subgroups to study the period required for dewaxing and clearing in 1,1,1 trichloroethane, then stained with H & E in the microwave oven at 360 W for 30 sec. Our conventional H & E staining procedure, which includes dewaxing, staining and clearing of sections, requires approximately 90 min, while our method using 1,1,1 trichloroethane and microwave heating required only 2 min. Our alternative method for H & E staining not only reduced the procedure time significantly, but also yielded staining quality equal or superior to those stained the conventional way. Our results suggest that 1,1,1 trichloroethane can be used effectively and safely as a dewaxing and clearing agent for H & E staining in a microwave oven.