Dmi8 microsystems

Immunofluorescence was performed using protocols described previously [27 ]. Mammary cells (2 × 10⁴ cells/well) were plated onto 12-well plates, fixed with 4% paraformaldehyde for 15 min, then washed 3 times with PBS and incubated with 0.3% or 0.5% Triton X-100 for 15 min at room temperature to increase the permeability. Cells were washed three times with PBS, incubated for 1 h with 5% BSA at 37 °C, and then incubated at 4 °C overnight with primary antibody (the same as that used in the Western blot analysis) in PBS containing 1% BSA and 0.3 Triton X-100 (T9284, Sigma-Aldrich). After PBS washes, cells were stained for 1 h with FITC labeled goat anti-rabbit FITC secondary antibody in a dark 37 °C room and then washed 3 times with PBS. DAPI (1 μg/mL) (D8417, Sigma-Aldrich) was used for nuclear counterstaining for 5 min, and then cells were washed 3 times. Cells were imaged using a DMi8 Microsystems GmbH (Leica, Wetzlar, Germany).

To confirm the co-expression of CD36 and CD8 on tumor infiltrating T lymphocytes, dual immunofluorescence was performed first, and the infiltration of other immune cells was explored by immunohistochemistry later. Tissue microarray construction and the IHC protocol have been described previously [12 (link)]. The details of the antibodies used in IHC assay are provided in Supplementary Table 3. IHC sections were scanned by Olympus CDD camera, Nikon eclipse Ti-S microscope (200×magnification) and NIS-Elements F3.2 software. Two experienced pathologists, blinded to the follow-up information, counted the number of positive staining cells at 200× magnification, and the average number was used as the final data. For IF staining, the slides were incubated with specific antibodies at 4 °C overnight. Then, samples were incubated with species-appropriate rabbit/mouse secondary antibodies coupled to Alexa Fluor dyes (488 (ab185033),594 (ab203419)) for 1 h at room temperature. DAPI (ab285390) containing anti-fluorescence quenching was used to mount cover slips, and the sections were analyzed through Leica DMi8 microsystems.

We used PBS to wash cells and then 4% paraformaldehyde to fix cells for 20 min after stretching. Triton X-100 (0.1%, Solarbio, Beijing, China) was used to permeate the cytomembrane. Cells were subsequently blocked with goat serum (10%, Solarbio, Beijing, China) for 1 h. Then, the primary antibodies were incubated with cells overnight at 4 °C. The secondary antibodies were incubated with cells for 1 h at room temperature after washing twice. DAPI was used to stain the cell nucleus. For 63× microscopy images, silicone membranes were cut from Bioflex culture plates and transferred to glass slides, and sealed with mounting medium (Abcam, Cambridge, UK). The Leica DMi8 microsystems (Lecia, Wetzlar, Germany) were utilized to visualize and picture cells. Semi-quantification analysis was implemented by the ImageJ software.

The cells after stretching were washed with PBS and fixed with 4% paraformaldehyde. To stain the shape of PDLSCs, 0.1% Crystal Violet Stain Solution (Solarbio, Beijing, China) was used. The Leica DMi8 microsystems (Lecia, Wetzlar, Germany) were utilized to image cells. Five random fields were selected per well, and ten cells were randomly chosen in every field to measure and analyze. Cellular phenotyping data including cell area, roundness, and width-to-length ratio were determined and analyzed using FIJI (NIH, USA) by thresholding.