Zen microscopy software

The survival of bacteria in hemolymph was examined using the LIVE/DEAD BacLight kit (Molecular Probes). The hemolymph was collected as described above and cytospun on polylysine glass. The staining procedure was performed according to the manufacturer’s instructions. Bacteria were visualized by confocal laser scanning microscopy (CLSM) using a Zeiss LSM 880 confocal system equipped with 100× oil immersion objectives. The acquired Z-stack images from five different fields of view were analyzed using Zeiss ZEN microscopy software and Fiji software. The quantification of bacteria in the hemolymph of G. mellonella was calculated as an area of fluorescence spot intensity using ImageJ software.

Four-month-old male mice were terminated, and their brains were dissected. Fixation, preparation of histological sections, staining with anti-tyrosine hydroxylase antibody (TH, mouse monoclonal antibody, clone TH-2, Sigma diluted 1:1000) and stereological counting of TH-positive neurons in the SNpc and ventral tegmental area (VTA) were performed as described [97 (link),98 (link)].
Briefly, the margins of SNpc and VTA on stained sections were outlined using distribution atlas of TH-positive cells [99 (link)]. The first section for counting was randomly chosen from the first ten sections that included the SN/VTA region. Starting from this section, on every fifth section, TH-positive cells with a clearly visible nucleus were counted through the whole region. ZEN Microscopy Software (Carl Zeiss) was employed to measure diameters of 30 nuclei of dopaminergic neurons in each of these regions of every mouse brain included in this study. The nuclei were chosen randomly, and the distance measured as the horizontal length as they appeared on the screen. A mean was calculated for each animal and used for Abercrombie’s correction [100 (link)] to obtain an actual number of TH positive cells in the structure.

The expression of C. parvum GP40 and GP15Δgpi in T. gondii was initially verified by immunofluorescence assay (IFA). For the IFA analysis of the RHΔku80 Tguprt-Cpgp40 mutant and the Tguprt-Cpgp15Δgpi mutant, HFF cells were infected with 10⁵ tachyzoites of these two mutants, respectively. After 16 h of culture at 37 °C with 5% CO₂, the HFF cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 at 37 °C for 10 min, and blocked with 1% BSA at 37 °C for 20 min. The following primary antibodies were diluted in 0.1% BSA for staining: mouse anti-HA (MBL Beijing Biotech, Beijing, China) at a 1:500 dilution, mouse anti-GP60 (purified rabbit pAbs) at 1:800 dilution. Cells were incubated with the antibodies for 40 min at 37 °C, followed by six washes with PBS. Alexa Fluor-conjugated secondary antibodies (Thermo) diluted 1:1000 and Hochest diluted 1:2000 in 0.1% BSA were added to the coverslips and incubated at 37 °C for 20 min. The cells were then washed six times with PBS and mounted on slides using Vectashield. The stained slides were examined using a Zeiss Axioskop Mot 2 fluorescence microscope (Carl Zeiss, Oberkochen, Germany). Images were manipulated using ZEN microscopy software (Carl Zeiss, Oberkochen, Germany).

To investigate the subcellular localization of SGD1, the CDS of SGD1 was cloned into the pCAMBIA1305-eGFP vector, and then transferred to the p19 containing Agrobacterium strain GV3101 (pSoup-p19). The ER marker 35S::HDEL-mcherry was co-expressed with 35S::SGD1-GFP in N. benthamiana leaves by agroinfiltration. To test whether SiUBC32 colocalized with SGD1, the CDS of SiUBC32 was inserted into the pBI121-mcherry vector. The 35S::SGD1-GFP and 35S::SiUBC32-mcherry vectors were co-transfected into foxtail millet protoplasts using PEG-mediated transfection^{19 (link)}. Sections were imaged on a confocal microscope (LSM700, Zeiss, Germany). Images were analyzed and processed by ZEN Microscopy Software (Zeiss, Germany). The primers used for subcellular localization assay are listed in Supplementary Table 6.

To determine the insulin content in pancreatic β cells, paraffin-embedded tissues were cut (4 µm) and these sections were incubated overnight at 4 °C with a 1:1000 dilution of rabbit monoclonal antibody against rat insulin (Cell Signaling Technology, Danvers, MA, USA). Detection was performed with a secondary antibody provided in the Mouse/Rabbit ImmunoDetector HRP/DAB Detection System (BIO SB, Hercules, CA, USA). Subsequently, the sections were counterstained with H&E. For negative controls, the primary antibodies were replaced with 1X PBS. The images of the pancreas slides immunostained with the antibody against insulin were analyzed using ZEN microscopy software (version 3.2, Carl Zeiss, Jena, Germany). A pathologist skilled in pancreatic morphometric analysis performed the semiquantitative interpretation, reading through the whole sample in the slide and assigning percentages to each staining degree and intensity of insulin-positive cells in the Langerhans islets. The identities of the samples from the control and the experimental groups were kept unknown to the pathologist during this analysis.