Confocal microscopy

Cellular immunofluorescence is utilized to detect the expression of tumor stem cell biomarker protein levels. The experimental steps are briefly described as follows:1 Cells were inoculated on coverslips, cultured overnight and washed three times.2 Cells were fixed with 4% paraformaldehyde at room temperature and permeabilized with 0.1% Triton-100. 3 Sealing of antigens at room temperature 1 hour.4 Primary antibody (CD44, EpCAM; Abclonal, China) was incubated overnight at four degrees and CY3-labeled secondary antibody(Abclonal, China) was added. Olympus confocal microscopy (Olympus, Japan) was used for photography.

The remaining part from each isolated iERM underwent immunohistochemical analysis. In brief, the surgically removed iERMs were placed immediately in 4% paraformaldehyde for at least 24 hours. Then iERMs were washed in PBS/0.1% TritonX-100 (PBST) two times, and penetrated in 0.1 M Glycin/PBS. After washing in PBST, iERMs were incubated in primary antibody (rabbit GFAP, 1:500, Cat.No.9269 (Sigma-Aldrich, Germany); mouse Nestin, 1:500, Cat.No.561230 (BD Pharmingen, Germany); rabbit Pax2, 1:200, Cat. No.2549-1 (Epitomics, Germany); goat Sox2, 1:500, Cat.No.sc-17320 (Santa Cruz, Germany); rabbit Ki-67, 1:1000, Cat.No.6013874 (Novocastra, Germany); mouse CRALBP, 1:500, Cat.No.sc-376082 (Saanta Cruz, Germany)) or blocking solution (Control) for 24 hours at 4°C, followed by washing and incubation with secondary antibodies (rabbit Alexa Fluor ® 488, 1:250, Cat.No.A21206 (Invitrogen, Germany); goat Cy3, 1:250, Cat.No. 705-165-147 (Dianova, Germany); mouse Cy5, 1:250, Cat.No.715-175-150 (Jackson Immuno, Germany)) for 1 hour at room temperature. The samples were then incubated in DAPI for 20 minutes and placed on the slides with mounting medium. All the immunofluorescence pictures (single plane images and Z-stacks) were taken by Olympus confocal microscopy (Olympus, Hamburg, Germany) and analyzed by a FluoView software (Olympus). Below is a list of all antibodies used and their dilution.

To evaluate the cytotoxicity of Rdots on cells, SKBR3 cells were incubated with 1 nM endocytosis beads for 6 h and then mixed with 10 nM Rdots for surface protein labeling. Cell viability was studied using Live/Dead cell double staining by incubating with 2 µM calcein-AM (Invitrogen, C3099) and 2.5 µM propidium iodide (PI, Sigma-Aldrich, P4864) for 30 min at 37 °C. Fluorescent images were acquired by Olympus confocal microscopy prior to viable/dead cell counting.

To detect BmUGT expression, we plated the B. mori cell line BmN-SWU1 in 6-well culture plates (10⁵ cells/well) and challenged with N. bombycis (spore: cell, 10:1). Then, 72 h after infection, infected cells were fixed in 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 for 5 min. The cells were subsequently blocked in PBS that contained 10% (w/v) goat serum and 0.5% (v/v) BSA for 1 h and incubated with anti-BmUGT (1:1000) for 1 h. Alexa488 was used to label the primary antibodies and DNA was stained with DAPI (Sigma, St. Louis, MO, USA) for 30 min. Fluorescence was observed and imaged with confocal microscopy (Olympus, Tokyo, Japan).

Both ends of the mice’s tibias/femurs were removed. Then, they were embedded in OCT for frozen sectioning and cut parallel to the long axis of the long bones. Stop cutting when the maximum cross section of the long bones was observed. The OCT around the rest of the bones were melted at room temperature. The remaining bone samples were washed three times in PBS for 10 min and fixed in 4% PFA for 2 hr. Then, they were immersed in 0.1% Triton X-100 for 1 hr, blocked using 3% BSA, and stained using Alexa Fluor 568 Phalloidin (Invitrogen) for 48 hr at 4°C in the dark with gentle shake. The samples were washed three times with PBS for 10 min. The cross section of the sample was inverted in the confocal dish. Pictures were captured using confocal microscopy (Olympus), and ImageJ was used to measure the number of dendrites per osteocyte.

Samples collected at the different stages of growth (mid-exponential, early and late stationary phases) were fixed using a 10% solution of Nigrosin (Sigma-Aldrich Chemie GmbH) and analysed by confocal microscopy (Olympus Life Science, Hamburg, Germany). For each growth phase and growth condition, the images were analyzed using the cellSens Entry software (v1.16) (Olympus Life Science) to determine the cell size of a minimum of 50 individual cells. All data points were then merged (per growth condition) to calculate an averaged cell length and the respective standard error of the mean (SEM) in GraphPad Prism (v8.1.1, GraphPad Software Inc.).

The expression of EBY100-OMCP and EBY100-LTB-OMCP was induced by galactose in the dark. A total of 1 mL of yeast (OD_600nm = 1) was harvested by centrifugation (4 °C, 3000× g, 5 min). The yeast cells were washed three times with sterile phosphate-buffered saline (PBS; Hyclone, Logan, UT, USA). After centrifugation (4 °C, 3000× g, 5 min), we added 500 μL murine 6 × His-tag antibody (1:1000) (ab18184, Abcam, Cambridge, UK) to the cells and incubated them at room temperature for 2 h. The cells were centrifuged again and washed three times with sterile PBS plus 0.05% Tween 20 (PBST). Then, 500 μL of goat anti-mouse IgG-H&L (Alexa Fluor 594) secondary antibody (1:500) (A-11005, Invitrogen) was added, and the cells were incubated in the dark for 1 h at room temperature. Yeast cells were washed three times with sterile PBST and resuspended in PBS. A small amount of the yeast suspension was coated on a sterile glass slide. Slides were subjected to confocal microscopy (Olympus, Tokyo, Japan) for imaging.