Bx41 compound microscope

Types are deposited in the Institute of Zoology, Chinese Academy of Sciences (IZCAS) in Beijing. All specimens collected were preserved and observed in a 95% ethanol solution. The specimens were measured and examined under a Leica M205C stereomicroscope, and further morphological details were observed using an Olympus BX41 compound microscope. The male palp was dissected from the left side of the spider for further examination. The carapace measurements include the clypeus (except for Relictocera sp. which has a distinct clypeus). The length and width ratios were measured according to the length of the cymbium (including the cymbial protrusion) to its width. The internal genitalia of the female and the male palp were dissected and immersed in lactic acid for digestion. An Olympus C7070 wide zoom digital camera (7.1 megapixels) mounted on an Olympus SZX12 stereomicroscope was used to take photos at different focal plans. The photos were assembled with the image stacking software Helicon Focus 6.7.1 to generate high quality photos before further editing with Adobe Photoshop CC 2014. Leg measurements are given as total length (femur, patella, tibia, metatarsus, and tarsus). Leg segments were measured from their retrolateral side. All measurements are given in millimetres (mm). Terminology follows Li et al. (2014) (link), Tong and Li (2007) , and Deeleman-Reinhold (1995) .

We used the acridine orange-ethidium bromide (AO-EB) dual-staining method to differentiate between condensed apoptotic or necrotic nuclei and normal cells. Briefly, HeLa cells were seeded on a coverslip-loaded 6-well plate at a density of 1 × 10⁵ cells/well and allowed to grow overnight. Then, the cells were exposed to 0.5 and 0.75 µg/mL of compound 1 for 24 h, washed twice with PBS to remove the remaining medium, and stained with equal volumes of AO and EB (20 μg/mL in PBS). Finally, the stained cells were washed with PBS and mounted onto a microscope slide in mounting medium, and images were obtained using appropriate filter settings in an Olympus BX41 compound microscope (Olympus) fitted with a fluorescence attachment and CCD camera. We also quantified apoptotic and necrotic cells on the basis of the uptake of AO and EB in more than 300 cells. The criteria for identification were as follows: a green intact nucleus and viable cells; dense green areas of chromatin condensation in the nucleus and apoptosis; and an orange intact nucleus and necrosis.

Types are deposited in the Institute of Zoology, Chinese Academy of Sciences (IZCAS) in Beijing. All specimens collected were studied and preserved in 95% ethanol. The specimens were measured and examined with a Leica M205 C stereomicroscope, and further morphological details were observed with an Olympus BX41 compound microscope. Male palps were detached from the left side of the animal for further examination (except for Leclerceraxiangbabang sp. nov. whose right palp was detached). Carapace length was measured excluding the clypeus. Internal genitalia of the female and palpal bulbs were dissected and immersed in lactic acid. An Olympus C7070 wide zoom digital camera (7.1 megapixels) mounted on an Olympus SZX12 stereomicroscope was used to take photos at different focal planes. The photos were then transferred to the image stacking software Helicon Focus 6.7.1 to generate photos with a greater depth of field before further processing with Adobe Photoshop CC 2014. Leg measurements are shown as total length: femur, patella, tibia, metatarsus, and tarsus. Leg segments were measured from their retrolateral side. All measurements are given in millimetres (mm). All terminology follows that of Li et al. (2014) (link).

Larval skeletal samples (phf21aa and kdm1a) were prepared and stained using Alcian Blue and Alizarin Red dyes as described (Walker & Kimmel, 2007 (link); DeLaurier, Alvarez & Wiggins, 2019 (link)). Samples were flat-mounted and imaged using an Olympus BX41 compound microscope and Olympus cellSens Standard software (version 1.16).

To detect the early stages of apoptosis, we measured the mitochondrial membrane potential (MMP) in treated and control HeLa cells by staining with a fluorescent probe, rhodamine 123. Briefly, HeLa cells were seeded on a coverslip-loaded 6-well plate at a density of 1 × 10⁵ cells/well, allowed to adhere for 24 h, then treated with 0.5 and 0.75 µg/mL of compound 1, and further incubated for 24 h. Next, the cells were washed with PBS (pH 7.4), fixed with ice-cold 70% ethanol, and incubated with 5 µg/mL of rhodamine 123 for 30 min at 37 °C. Finally, the cells were again washed with PBS and mounted onto a microscope slide in mounting medium, and images were obtained using appropriate filter settings in an Olympus BX41 compound microscope (Olympus) at 400× magnification.

All specimens were collected in China, Thailand and Laos (Fig. 22), and preserved in 95% ethanol. Types of all new species are deposited in the Institute of Zoology, Chinese Academy of Sciences in Beijing, China. Specimens were examined and measured using a Leica M205 C stereomicroscope. Morphological details were studied with an Olympus BX41 compound microscope. Photos were taken with an Olympus C7070 wide zoom digital camera (7.1 megapixels) mounted on an Olympus SZX12 stereomicroscope. The images were montaged using Helicon Focus 6.7.1 image stacking software. The map was generated using ArcView GIS 10.2. All measurements are in millimeters (mm). Leg measurements are shown as total length (femur, patella, tibia, metatarsus, and tarsus). Leg segments were measured from the retrolateral side. Carapace length was measured from the anterior eye row to the carapace posterior margin. Terminology follows that of Deeleman-Reinhold (1995) , Tong and Li (2007) , and Li et al. (2014) (link).