Dm4 b upright microscope

The examined specimens were collected from Guangxi and Fujian Provinces in southeastern China in 2018. The descriptive terminology of the anatomical structures generally follows Wang (2006a) , however in descriptions of the male genitalia, the more proper term phallus rather than aedeagus is applied here following Kristensen (2003) . Photographs of adults were taken using a Canon EOS 6D Mark II camera plus an EF 100 mm f/2.8L MACRO IS USM lens with the help of EOS Utility 3.10.20 software. Images of genitalia were captured using a Leica DM4 B upright microscope and photomontage was performed with Leica Application Suite X imaging software. All type specimens are deposited in the Morphological Laboratory, Guizhou University of traditional Chinese Medicine, Guiyang 550025, Guizhou, China.

All specimens for this study were collected in 2018 from the Hainan and Hubei provinces of China. The descriptive terminology of the anatomical structures follows Wang (2006) , Yin et al. (2014) (link) and Kristensen (2003) (link). Photographs of adults were taken using a Canon EOS 6D Mark II camera with an EF 100 mm f/2.8L MACRO IS USM lens assisted by the EOS Utility 3.10.20 software. Stacked images of the genitalia were captured using a Leica DM4 B upright microscope. Photomontage was performed with the Leica Application Suite X imaging software. Species distribution data were compiled within Microsoft Excel using both published records and specimen label data. The distribution map was produced with the aid of DIVA-GIS 7.5 (Hijmans et al. 2011 ).
All type specimens are deposited in the Morphological Laboratory, Guizhou University of Traditional Chinese Medicine, Guiyang, Guizhou, China.

CAH gel was first degraded by sodium citrate (50 mg/ml) and type I collagenase (1 mg/ml), and organoids were digested by Accutase (Thermo Fisher Scientific) to obtain single cell suspension. About 1 × 10⁴ resuspended cells in 200 μl of PBS were loaded in the cytospin chamber; cell suspension was centrifuged at 500 g for 5 min by using Cytospin®4 Cytocentrifuge (Thermo Fisher Scientific); and then the cells were collected on glass slides for subsequent IF staining experiment which was described in the above section. Images of samples stained with specific markers for ciliated cells (βIV-Tubulin) and goblet cells (Mucin 5AC) were captured at 200x or 400x magnification under DM4B upright microscope (Leica). The βIV-Tubulin⁺ or Mucin 5AC⁺ cells were counted in three randomly selected areas (200x magnification) and the percentages of ciliated cells and goblets cells among total epithelial cells (more than 300 cells totally) were calculated.

After 30 days of salinity stress, the gill tissues of grass carp in the untreated control group and gill tissues from symptomatic and asymptomatic grass carp in 3‰ and 6‰ salinity-treated groups were taken and fixed in 4% paraformaldehyde solution at 4°C for 24h. The fixed gill tissues were processed through the conventional paraffin embedding technique, and sliced into 5 μm-thick tissue sections. Then the slides from 3 fish per group were subjected to Hematoxylin and eosin staining, sealed with neutral gum, observed and photographed with a Leica DM4 B upright microscope.

A section of the aorta was dissected from each rat, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned at 5 μm, and mounted on slides. The slides were transferred to an oven and baked for 30 min at 60°C to melt the wax, which was then removed by treatment with xylene for 20 min. Tissue sections were rehydrated in a descending ethanol series of 100% for 5 min and 95%, 90%, 80%, and 70% for 3 min each. After washing three times in PBS, the sections were stained with hematoxylin and eosin or incubated with a primary antibody. All images were captured with a Leica DM4B upright microscope (Leica Inc., Germany).

IF was performed on paraffin embedded sections (5 µm) as previously described 23 . Briefly, sections were deparaffinized in xylene and ethanol before antigen retrieval in sodium citrate buffer (10 mM, pH 7.5) for 20 min at 90 ° C. Cultured cells were plated on coverslips, cultured overnight and treated the next day. At collection cells were fixed in 4% paraformaldehyde and permeabilized in 0.5% Triton X-100/PBS. Acrolein antibody (Invitrogen MA527553 Waltham, MA) was used at 1:50 and anti-4-Hydroxynonenal antibody (ab46545 from Abcam Cambridge, MA) was used at 1:100 and incubated overnight at 4 ° C, anti-γH2A.X (Cell Signalling Rabbit mAb#9718) was used at 1:500. Secondary goat anti-rabbit Daylight 488 (Fisher PI35552) was used at 1:500 and incubated for 45 min in the dark at room temperature. Following 3 washes, slides were then mounted with DAPI mounting media. Images were captured using the Leica DM4 B upright microscope (Leica Chicago, IL).