Immunostaining permeabilization buffer with triton x 100

Cell proliferation was monitored using BeyoClick™ EdU-555 (Beyotime Biotechnology, C0075L) according to the manufacturer’s instructions. Briefly, 1 × 105 cells were cultured in six-well plates with 10 mM EdU containing complete medium for 2 h and fixed with Immunol Staining Fix Solution (Beyotime Biotechnology, P0098) at room temperature for 15 minutes. Then, the cells were permeabilized with Immunostaining Permeabilization Buffer with Triton X-100 (Beyotime Biotechnology, C0075L) at room temperature for 15 minutes. Subsequently, 500 μL Click Additive Solution (Click Reaction Buffer: 430 μL, CuSO₄: 20 μL, azide 555: 1 μL, and Click Additive Solution 50 μL) and Hoechst 33342 were added to the cell and incubated in the dark at room temperature for 30 minutes. Finally, cell proliferation was analyzed using FACSAria SORP (BD Biosciences, USA) and FlowJo program.

Immunofluorescence staining was performed as previously described [35 (link)]. Frozen brain sections (7 μm) or cultured cells on coverslips were fixed in 4% paraformaldehyde for 10 min. Then, they were treated with Immunostaining Permeabilization Buffer with Triton X-100 (Beyotime, Shanghai, China) for 30 min and were blocked with Immunol Staining Blocking Buffer (Beyotime, Shanghai, China) for 60 min. These samples were incubated with specific primary antibodies for ACSL4 (Affinity, DF12141, 1 : 200), FTH (Affinity, DF6278,1 : 200), GPX4 (Affinity, DF6701,1 : 200), NeuN (MilliporeSigma, MAB377X, 1 : 200), 8-hydroxyguanosine (8-OHdG) (Bioss, bs-1278R, 1 : 100), and SIRT1 (Santa Cruz, sc-74465, 1 : 50) in Universal Antibody Diluent (New Cell & Molecular, Suzhou, China) at 4°C overnight. Then, the samples were slowly washed three times with phosphate-buffered saline with 0.5% Tween-20 (PBST) and incubated with corresponding secondary antibodies (Cy™3-conjugated goat antirabbit IgG or goat antimouse IgG, 1 : 200) for 1 h at room temperature (RT). After washing 3 times with PBST, the samples were counterstained with 4,6-diamidino-2-phenylindole (DAPI, MilliporeSigma, 1 : 2000) for 10 min at RT. Fluorescence was visualized by a fluorescence microscope (ZEISS, Scope A1, Germany).

To explore the apoptotic in QBC939 and RBE cells, apoptosis was examined using One Step TUNEL Apoptosis Assay Kit (Beyotime, Beijing, China). Transfected cells were washed by PBS after fixed with paraformaldehyde for 30 min. Then, the cells were incubated by Immunostaining Permeabilization Buffer with Triton X-100 (Beyotime, Beijing, China). Afterwards, QBC939 and RBE cells transfected with si-NC or si-HOTAIR were incubated at 37 °C for 60 min after treated with 45 μl fluorescent labeled reagent and 5 μl terminal deoxynucleotidyl transferase (TdT). A fluorescent microscope (Leica, Germany) was used to detect the apoptotic cells.

One Step TUNEL Apoptosis Assay Kit (Beyotime, Beijing, China) was used to detect cell apoptosis. Treated cells were fixed with paraformaldehyde for 30 min. After washing with PBS, cells were incubated with Immunostaining Permeabilization Buffer with Triton X-100 (Beyotime, Beijing, China). Afterwards, cells were treated with 45 μl fluorescent labeled reagent and 5 μl terminal deoxynucleotidyl transferase (TdT) before incubated at 37°C for 60 min. Cells were observed by a fluorescent microscope (Leica, Germany).

First, 2 × 10⁵ 4T1 tumor cells were seeded into the CLSM-specific culture disk and then cultured for 24 h. Second, after replacing the culture medium with 1 mL of RPMI-1640 complete medium containing Au@Cu_2-xSe (50 µg mL⁻¹) or H₂O₂ (100 µM), the cells were irradiated with or without NIR laser (1.0 W cm⁻²) for 5 min. After irradiation, the cells continued to incubate for 6 h. Third, these cells were fixed with 4% paraformaldehyde for 10 min, washed several times with PBS, permeabilized with immunostaining permeabilization buffer with triton X-100 (Beyotime Biotechnology) for 10 min, and blocked with QuickBlock blocking buffer (Beyotime Biotechnology) for 10 min at room temperature. Forth, after washing three times with PBS, the cells were stained with anti-gamma H2AX (phospho S139) antibody [EP854(2)Y] (Abcam, dilution 1:400) at 4 °C overnight. Fifth, after washing three times with PBS, the cells were incubated with the goat polyclonal secondary antibody to rabbit IgG-H&L (Alexa Fluor® 647) (Abcam, dilution 1:400) for 1 h. Sixth, after washing three times with PBS, the cells were stained with DAPI (Beyotime Biotechnology) for 5 min. Finally, the cells were washed three times with PBS followed by the addition of 1 mL PBS. Then the fluorescence images of cells were collected by CLSM.

Human pancreatic tissue samples were collected from patients of RuiJin Hospital, Shanghai Jiaotong University, and the experimental protocols have been approved by the Ethics Committee of RuiJin Hospital, Shanghai Jiaotong University (Approval number: 2021–194). Tissues were fixed with 4% paraformaldehyde, then embedded and sliced in paraffin. The sections were dewaxed and heated in the Improved Citrate Antigen Retrieval Solution (Beyotime Biotechnology, P0083) for antigen repair and permeabilized with Immunostaining Permeabilization Buffer with Triton X-100(Beyotime Biotechnology, P0096). Endogenous peroxidase was blocked with Endogenous Peroxidase Blocking Buffer (Beyotime Biotechnology, P0100A). The sections were incubated with mouse anti-CD163 antibody (Cell Signaling Technology, 1:400) or anti-LDHA antibody (Cell Signaling Technology, 1:400). Antibody binding was detected with HRP-labeled secondary antibody (Santa Cruz Biotechnology Inc.) and visualized by the DAB+ Substrate Chromogen System (Dako Omnis). Samples were counterstained with hematoxylin, incubated in ethanol and xylene solution of ascending concentration, and finally fixed.