Ds ri2

We have tried to use Confocal Laser Scanning Microscopy and MicroXCT even SRμCT to observe more morphological characters, but with no success. It might be caused by the fact that these insects are too small and nearly semitransparent. Photographs were taken by using a Nikon SMZ 25 microscope with a Nikon DS-Ri 2 digital camera system, and the enlarged images of details of the specimens were taken using a Nikon ECLIPSE Ni microscope with a Nikon DS-Ri 2 digital camera system. Photographs with green background were taken using green epifluorescence as the light source attached to Leica DM5500B with an ANDOR Zyla digital camera system (Fig. 3d). Line drawings were prepared by using Adobe Illustrator CC and Adobe Photoshop CC graphics software.

The material described here is housed in the Key Lab of Insect Evolution and Environmental Changes, College of Life Sciences, Capital Normal University, Beijing, China (CNUB; Dong Ren, Curator). The specimen CNU-PHA-NN2019006 was examined under a Leica M205C dissecting microscope. All photographs were taken with a Nikon SMZ 25 microscope with an attached Nikon DS-Ri2 digital camera system and a Nikon ECLIPSE Ni microscope with an attached Nikon DS-Ri2 digital camera system. Line drawings were prepared using Adobe Illustrator CC and Adobe Photoshop CC graphics software.
The wing-venation nomenclature follows Wang et al. (2014) [44 (link)]. The following abbreviations have been used throughout: 1A, the first anal vein; Cu, cubitus; CuA, cubital anterior; CuP, cubital posterior; CuPa, anterior branch of CuP; CuPaα, anterior branch of CuPa; CuPaβ, posterior branch of CuPa; CuPb, posterior branch of CuP; M, media; MA, medial anterior; MP, medial posterior; R, radius; RA, radial anterior; RP, radial posterior; ScA, subcostal anterior; ScP, subcostal posterior.

Whole fruit and pericarp surfaces were examined using a binocular dissection scope (Nikon, SMZ 1270) and imaged with an attached camera (NIKON, DS-Ri2). Samples were stained with the water-soluble Safranin-O dye (Sigma-Aldrich) to examine xylem transport towards the fruit. Six strands were sampled at each time point along the course of fruit development. Three of the strands were placed for 4 h in 5 ml 1% Safranin-O solution and three were placed in 5 ml deionized water as a control. To monitor Safranin-O transport along the xylem and into the fruit pericarp tissue, a longitudinal section of the fruit (through the attached strand segment and at the center of the calyx to the distal end, the stigma trace) and a cross-section (at the middle between the calyx and the distal end) were generated and photographed using zoom stereomicroscope (SMZ1270, Nikon) equipped with a camera (DS-Ri2, Nikon). Fresh lemon juice was applied to avoid oxidation and pericarp browning as a result of the cut.

Cell growth on the surface of the planer-shaped PC(PEW-C₁₂E₅)-gel was observed. The planar-shaped PC(PEW-C₁₂E₅)-gel (20 × 15 × 1 mm) was washed as described above using a 40/60 ethanol/PBS ratio. The washed gel was placed in a 3.5 cm Petri dish. Three milliliters of NCI-H460 cells suspended in RPMI 1640 and 10% FBS medium 2 × 10⁵ cells mL⁻¹ density was seeded in the dish and cultured in a CO₂ incubator at 37 °C with 5% CO₂ for 96 h. The culture medium was replaced with fresh medium at the time of 48 h. The same experiment was performed without the gels, and cells were cultured on a tissue culture-treated culture dish (Corning Inc., #430165). Cells were observed at 48 and 96 h using a microscope (Eclipse Ti2-U, Nikon, Japan, CCD camera DS-Ri2, Nikon, Japan). 96 h-cultivation cells were stained with -Cellstain- Double Staining Kit (Dojindo, Inc., Kumamoto, Japan), and the living cells were observed using a fluorescent microscope (Eclipse Ti2-U, Nikon, Japan, CCD camera DS-Ri2, Nikon, Japan).

For immunohistochemistry, after re-hydration, tissue sections were incubated with Tris-HCl 20 mM, EDTA 0.65 mM, Tween-20 0.05% pH 9, in a microwave for 5 min (two times) for antigen retrieval. Sections were treated with H₂O₂ for 20 min, washed, incubated for 1 h at room temperature with 5% bovine serum albumin (BSA) and 0.3% Triton X-100 in PBS, and then overnight at 4 °C with anti-TRPML-1 Ab. Thereafter, slides were incubated for 30 min at room temperature with a biotinylated secondary Ab, rinsed, and exposed for 30 min to the streptavidin–biotin complex (VECTASTAIN ABC Kit, Vector laboratories, Burlingame, CA, USA). Immunoreactivity was detected by the addition of 3,3′-diaminobenzidine (DAB, Peroxidase Substrate Kit, Vector laboratories, Burlingame, CA, USA). Four random fields of each tissue specimen were analyzed under ×40 magnification using the Leica DMR Microscope collected by TV camera (Nikon DS-Ri2) with an NIS Element Imaging Software (Nikon Instruments, Firenze, Italy).
Untransfected, siGLO, siTRPML-1, pCMV, and pCMV-pTRPML-1 T98 and U251 cells were maintained on 8-well culture slides in fresh medium. After 72 h, cells were fixed with 2% and 4% paraformaldehyde with 0.5% of Triton X-100 in PBS for 10 min at room temperature and were then counterstained with hematoxylin. Immunohistochemistry in these slides was performed as described above, without antigen-retrieval.

For histological analysis, fixed lymph nodes were dehydrated in 100% ethanol and embedded in paraffin (Leica) using a Leica EG1150 tissue embedder. 7-μm-thick sections using a HM340E Thermo Scientific microtome were processed for hematoxylin-eosin (HE) (Leica) and immunohistochemistry staining as described (41 (link)). For immunohistochemistry of histology slides the following antibodies were used: anti-TER-119 (R&D Systems, MAB1125) and Alexa Fluor 488 conjugated goat anti-rat IgG antibody (Life Technologies A11006). Stained slides were mounted with Vectashield DAPI Mounting Medium (Vector Laboratories, H-1200). Tissue section samples were imaged using a Nikon Ni-U upright microscope (Nikon Instruments) using a 40x dry objective connected to a Nikon DS-Ri2 camera.

In situ hybridization images were taken on either a Zeiss AxioScop 2 mounted with an Axiocam camera triggered by Axiovision software (Carl Zeiss), a Zeiss Axio Imager A2 mounted with a Canon 6D triggered by Canon professional software, or a Nikon NTi using a Nikon DS-Ri2 color camera and the Elements software (Nikon). All expression patterns described here have been submitted to Kahi Kai, a comparative invertebrate gene expression database [62 (link)] hosted at http://www.kahikai.org/index.php?content=genes. Scoring of treatment phenotypes was performed on either a Zeiss Z-1 Axio imager or a Zeiss Axio Imager A2 microscope and confocal imaging was conducted on either a Zeiss LSM710 or Zeiss LSM Exciter microscope running the LSM ZEN software (Carl Zeiss). Fluorescent images were false-colored. The fluorescent channels were merged using ImageJ (http://rsbweb.nih.gov/ij/) and cropped to final size in Photoshop Cs6 (Adobe Inc.). Confocal images for Fig. 5 were processed using Imaris 8.1 (Bitplane).