Linear measurements were taken as in Derkarabetian and Hedin (2014) using an Olympus SZX12 dissecting microscope with an ocular micrometer. For SEM imaging, genitalia were extended from the body by opening the genital and anal opercula, and inserting a small blunt insect pin through the anal operculum and pushing the genitalia out. Specimens were dried using a critical point dryer, mounted on Ted Pella aluminum SEM stubs using copper conductive tape and coated with a 0.6 nm platinum coat. Multiple coats were applied in order to ensure proper coverage and to prevent charging. Coated specimens were imaged on a Quanta 450 SEM at the San Diego State University Electron Microscope Facility. Additionally, genitalia were examined using an Olympus BX40 compound microscope with a drawing tube. Genitalia were dissected from the body as above, then cleared in 10% KOH before viewing.
Bx40 compound microscope
Manufactured by Olympus
Sourced in Japan
The BX40 is a compound microscope manufactured by Olympus. It features a sturdy, ergonomic design and is equipped with high-quality optics for clear and detailed observations. The BX40 is suitable for a wide range of applications in various fields of study.
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7 protocols using bx40 compound microscope
1
Genitalia Preparation and Imaging Protocol
2
Histological Analysis of Nodular Tissues
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Tissues containing nodules from each animal identified as positive (n = 27) were fixed in 10% formalin in seawater (Howard et al. 2004 ). Samples of fixed tissue were embedded in paraffin, sectioned, and stained with haematoxylin and eosin. Sections from all animals were stained with Ziehl-Neelsen acid-fast stain and selected cases were stained with Brown and Brenn Gram stain (Mass Histology Service) (Howard et al. 2004 ). Stained sections were evaluated for location, appearance and staining characteristics on an Olympus BX40 compound microscope with an attached Olympus DP 25 camera.
3
Rapid Golgi Staining Techniques
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This manuscript primarily relies on the results of rapid Golgi studies carried out over a period of years. The method requires “fine tuning” not only for species but also other important factors such as age, brain area, and cell type, and even so, the technique is “finicky” (refer to Scheibel and Scheibel, 1970 (link); Valverde, 1970 (link)). Thus, repeated trials with variations of staining procedure were necessary to reveal the four major cell types identified in the TRP. The basic rapid Golgi method of Valverde (1970) (link) with some modifications by Morest and Morest (1966) (link) was used (see Evan et al., 1976 (link)). Modifications for SF brains include slight variations of immersion fixation and “staining” times. Tissue was embedded in celloidin and sections were cut between 100 and 150 μm in all three planes using an American Optics sliding microtome (model 860-now under Reichert Inc., Depew NY, United States) (Kiernan, 2015 ). Using a Wild M20 compound microscope (Heerbrugg, Switzerland), areas of interest were traced using 20–100 magnification eyepieces with a camera lucida or later a video screen. Stained cells were photographed using an Olympus BX40 compound microscope and DP71 camera system (Olympus scientific, Waltham, MA, United States). This Golgi study entailed the use of 38 brains (20 transverse, 8 horizontal, and 10 sagittal series).
4
Digenean Parasite Collection Protocol
5
Macromorphological and Microscopic Analysis
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Specimens studied are deposited at the Farlow Herbarium of Harvard University (FH), MA, USA, Beijing Forestry University (BJFC), Beijing, China and United States National Fungus Collections (BPI), New York, NY, USA. Macromorphological descriptions were on the basis of field notes and study of specimens. Color terms followed previous studies [29] . Microscopic measurements were made from slide preparations of dried specimens stained with Cotton Blue and Melzer's reagent by light microscopy [30, (link)31] (link). Sections were studied using an Olympus BX40 compound microscope (Tokyo, Japan). In presenting spore size variation, 5% of measurements were excluded from each end of the range. The following abbreviations are used: KOH = 5% potassium hydroxide, CB = Cotton Blue, CB+ = cyanophilous; IKI = Melzer's reagent, IKI-= both non-amyloid and non-dextrinoid, L = mean spore length (arithmetic average of all spores), W = mean spore width (arithmetic average of all spores), Q = mean each spore length/width ratio, n (a/b) = number of spores (a) measured from given number of specimens (b).
6
Ovarian Diapause Identification Protocol
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Ovaries were dissected from females aged 6–8 days in 1% PBS solution and mounted with Aqua-Poly/Mount (Polysciences Inc). To confirm reproductive diapause, primary follicle lengths were measured under ×200 magnification using an Olympus BX40 compound microscope according to ovary morphology as previously defined28 (link),29 . Ten measurements per ovary were recorded to calculate an average follicle length per individual. Individuals with average follicle size under 50 μm were defined as being in diapause29 .
7
Histological Analysis of Nectary Development
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Pre-secretory and secretory stage nectaries were fixed for several days at 4°C, in a solution of 3% (w/v) glutaraldehyde and 2% (w/v) paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.2. Samples were dehydrated in a graded ethanol series (50%–100%), followed by infiltration and embedding over 5 d in LR White resin. For replication purposes, a minimum of four nectaries per nectary type was imbedded at each developmental stage. Resin blocks were polymerized at 55°C for 72 h. Histological sections were cut at 1.3-µm thickness using a Leica UC6 Ultramicrotome (www.leica-microsystems.com ). Sections were dyed with toluidine blue O for general contrast and PAS technique for starch and other non-water-soluble carbohydrates (Ruzin, 1999 ). Digital images were collected using a Zeiss Axiocam HRC camera (www.zeiss.com ) on an Olympus BX-40 compound microscope (www.olympus-ims.com ) in bright-field mode.
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