Siliques of Arabidopsis plants expressing TCOp::TCO-GUS were GUS-stained for 24 h as previously detailed [58 (link)], fixed overnight in FAA (3.7% formaldehyde, 50% ethanol, 5% glacial acetic acid), embedded in paraffin, and sectioned to a thickness of 20 µm. Sections were then deparaffinized, rehydrated through a reverse ethanol series (100% to 30%), incubated in water, mounted and imaged. For embryo dissection, seeds were fixed in 4% paraformaldehyde under vacuum for 2 h, rinsed three times in phosphate buffered saline, and incubated in clearing solution (6M urea, 30% glycerol, 0.1% Triton X-100) [63 (link)] for 3 weeks at 4 °C. Embryos were liberated from the seed coat by applying gentle pressure. An Olympus SZX16 dissecting microscope was used to capture images of live plant tissues, while an Olympus BX61 compound microscope was used to capture images of fixed/sectioned tissues and GFP fluorescence (Olympus, Center Valley, PA, USA).
Bx61 compound microscope
Manufactured by Olympus
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The BX61 is a compound microscope from Olympus. It is designed for high-performance observation and imaging of specimens. The BX61 features high-quality optics, advanced illumination, and a sturdy construction to provide reliable performance for a variety of applications.
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11 protocols using bx61 compound microscope
1
Arabidopsis Silique Sectioning and Embryo Dissection
2
Mite Stylet Measurement Protocol
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For LM measurements, the mites were collected and stored in 80% ethanol and slide-mounted later in Hoyer’s medium (Walter and Krantz 2009 ). Micrographs were taken using the Olympus BX61 compound microscope. The stylets were measured using Figi measurement software (Schindelin et al. 2012 (link)).
For SEM measurements, mites that were observed feeding were sprayed with chloroform to attempt to quickly kill them while the stylets were protracted and then transferred into 80% ethanol to preserve them. In the laboratory, the mites were dehydrated further through 3 changes of 100% ethanol then critical point dried as above. Each mite was then carefully mounted on a stub, sputter coated and observed as above. The stylet images were measured using Figi measurement software (Schindelin et al. 2012 (link)).
For SEM measurements, mites that were observed feeding were sprayed with chloroform to attempt to quickly kill them while the stylets were protracted and then transferred into 80% ethanol to preserve them. In the laboratory, the mites were dehydrated further through 3 changes of 100% ethanol then critical point dried as above. Each mite was then carefully mounted on a stub, sputter coated and observed as above. The stylet images were measured using Figi measurement software (Schindelin et al. 2012 (link)).
3
Birefringence Microscopy of Embryo Tails
4
Transmitted and Polarized Light Imaging of Sugar Films
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Transmitted light images of the sugar films were collected using an Olympus BX-61 compound microscope, equipped with a 20x, 0.50 NA objective (UPlan FL N), and a tungsten-halogen light source. Image capture was via an Olympus DP72 color cooled CCD camera, at 2,048 × 1,536 pixels. Polarized light images were captured with this same configuration using the Olympus U-PO accessories. Macroscopic transmitted-light images of the sugar films were collected using an Olympus SZX-16 stereoscope, using a 1X, 0.15NA objective (SDFPLAPO1 x PF), and an Olympus DP71 cooled color CCD camera, at 2,048 × 1,536 pixels.
5
Taxonomic revision of the flea genus Phalacropsylla
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Specimens were obtained on loan from the following institutions: Brigham Young University flea collection, Monte L. Bean Life Science Museum, Brigham Young University, Provo, Utah, USA (BYUC ); The Carnegie Museum of Natural History, Pittsburgh, Pennsylvania, USA (CMNH ); Colección de Siphonatera, Museo de Zoología, Facultad de Ciencias, Universidad Nacional Autónoma de México, Ciudad de Mexico, Mexico (MZFC-S ); the Department of Entomology, National Museum of Natural History, Smithsonian Institution, Washington, D.C., USA (USNM ).
The most stable and representative characters for the genusPhalacropsylla Phalacropsylla BYUC was part of the Glenn E. Haas flea collection. A designated collector for most of the fleas from his collection was not indicated on his slides but were undoubtedly collected by him. The map was prepared with ESRI® ArcGIS version 10.5. Flea images were illustrated with the aid of an Olympus BX61 Compound Microscope and an Olympus CC12 digital camera accompanied with an Olympus Microsuite™ B3SV program.
The most stable and representative characters for the genus
6
Genetic Basis of GFP Mutants
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For allelism test, the mutants were crossed to each other, and GFP fluorescence in F1 seeds was compared to that in the wildtype parent line, using an Olympus BX61 compound microscope. To determine the genetic basis of the mutations, the mutants were crossed to wild type plants (Columbia). In the F2 segregating population, the number of mutants and normal plants with GFP fluorescence were counted, and the number of genes involved as well as dominance or recessiveness of the mutations were determined by χ² test.
7
EMS Mutagenesis of WOX7pro::GFP Arabidopsis
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Mutagenesis was conducted according to the Arabidopsis handbook [26 ]. Briefly, approximately 20,000 seeds homozygous for the WOX7pro::GFP reporter gene in the Columbia background were added to 10 mL 0.1% ethyl methanesulfonate (EMS, Sigma M0880, MO, USA) in a 15 mL conical tube, and the tube was rotated on a rotator overnight at room temperature. After thorough washing with sterile H2O, the seeds were suspended in 0.01% agarose and sowed in soil (three to five seeds per pot). At maturity, the seeds from all plants in a pot were pooled.
For mutant screening, 100 seeds from each tube were germinated in Murashige and Skoog medium (MS) medium. Seven days after germination, seedlings were transferred to a MS plate supplemented with 5% glucose and allowed to grow for one more day. For microscopic examination, roots were cut and loaded in H2O on a slide, and GFP fluorescence was checked on an Olympus BX61 compound microscope. As a control, seedlings with the same promoter-GFP construct without EMS treatment were grown in a similar manner.
For mutant screening, 100 seeds from each tube were germinated in Murashige and Skoog medium (MS) medium. Seven days after germination, seedlings were transferred to a MS plate supplemented with 5% glucose and allowed to grow for one more day. For microscopic examination, roots were cut and loaded in H2O on a slide, and GFP fluorescence was checked on an Olympus BX61 compound microscope. As a control, seedlings with the same promoter-GFP construct without EMS treatment were grown in a similar manner.
8
GUS Staining of Arabidopsis Seedlings
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GUS staining was performed essentially as described in the Arabidopsis book [26 ]. Seedlings were incubated in GUS staining solution overnight at 37 °C. For light microscopy, the roots were first cleared in a drop of chloral hydrate solution (7.5 g chloral hydrate dissolved in 3 mL 50% glycerol) on a glass slide for 1–5 min. Images were captured using an Olympus BX61 compound microscope.
9
Ectoparasites of Lesser Long-nosed Bats
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As part of a long-term ecological study of insectivorous bats in the southwestern United States between 23 June and 4 September 2013, 23 Lesser Long nosed bats, Leptonycteris yerbabuenae Martinez and Villa (Phyllostomidae ), were captured using a mist net placed adjacent to a hummingbird feeder. Bats were weighed, measured, and examined and fleas were removed with forceps and preserved in 70% ethanol pending processing. A total of three female fleas were collected from the ears of a young adult male Leptonycteris yerbabuenae . One flea is deposited in the Brigham Young University DNA flea voucher collection and the other two in the collection of Christopher Newport University. Images were prepared using an Olympus BX61 Compound Microscope, Olympus CC12 digital camera accompanied with an Olympus Microsuite™ B3SV program and Adobe Photoshop, CS4.
10
Immunohistochemical Staining of Extracellular Matrix
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Slides were washed in PBS 2 × 5 min. Antigen retrieval was then performed by microwaving slides in 1× epitope unmasking buffer (cat 21760005-1; Bioworld) for 2 min at a temperature just below 100℃ in a glass staining dish. Covered staining dish was then wrapped with a blue underpad and foil for 10 min at room temperature, placed at 4℃ for 20 min, then washed in PBS 2 × 5 min. Slides were placed in blocking solution consisting of 5% heatinactivated goat serum, 0.1% Triton (cat 100504-970; VWR), 0.02% SDS for 1 hr at room temperature. Slides were then incubated with primary antibodies anti-Procollagen I (cat SP1.D8; DSHB) 1:20, anti-collagen XIVA1 (Novus NBP1-86877) 1:250, or anti-periostin 1:100 (cat ab14041; Abcam) diluted in block solution overnight at 4℃. Slides were washed in PBS + 0.1% Triton 3 × 10 min. Slides were then incubated in the dark in Alexa Fluor secondary antibodies goat anti-mouse 488 (cat A21121; Life Tech) 1:500, goat anti-rabbit 594 (cat A11012; Life Tech) 1:500, and 1 µg/ml 4′,6-diamidino-2-phenylindole (DAPI) (cat D1306; Life Tech) diluted in block solution for 1 hr at room temperature. Slides were then washed in PBS + 0.1% Triton 3 × 10 min and PBS 1 × 5 min then coverslipped with Fluoromount G (cat OB10001; Southern Biotech) under glass coverslips. All images were taken on an Olympus BX61 compound microscope.
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