To examine the PD-L1 expression in the distal tumor tissues under varying treatments, the tumor tissues were isolated from each mouse in different treatment groups to perform immunohistochemistry assay. The collected tumors were placed in 4% paraformaldehyde for 6 to 8 h and transferred to 20% sucrose and the frozen tissue pieces were embedded. A frozen tissue section of 8 μm thick was prepared by a cryostat fixed with acetone for 10 min. The above sections were washed three times with PBS solution for 3 to 5 min each time. Subsequently, the sections were then blocked with 2% BSA at room temperature for 1 h. After incubation with PD-L1 primary antibody overnight at 4 ℃, the slices were washed three times with PBS. Subsequently, it was incubated with a secondary antibody at room temperature for 1 h, and washed with PBS. After staining with Hoechst (keygen) for 10 min, it was washed twice with PBS, an anti-quenching agent was added dropwise to seal, and it observed using a confocal scanning laser microscope (CSLM) (Olympus, FV1000).
Hoechst
Manufactured by Keygen Biotech
Sourced in China, Germany
The Hoechst is a laboratory equipment used for staining and visualizing DNA in biological samples. It functions by binding to the minor groove of double-stranded DNA, emitting a fluorescent signal that can be detected and analyzed.
Automatically generated - may contain errors
Lab products found in correlation
Fv1000, by Olympus (2 mentions)
Confocal microscope, by Zeiss (2 mentions)
α actinin antibody, by Merck Group (2 mentions)
Lysotracker green, by Keygen Biotech (1 mentions)
Ribonucleoside vanadyl complexes, by Merck Group (1 mentions)
μ slide 8 well chamber, by Ibidi (1 mentions)
Edu imaging kit, by Keygen Biotech (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Ki67 antibody, by Abcam (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Cd8 primary antibody, by Thermo Fisher Scientific (1 mentions)
6 protocols using hoechst
1
Quantifying PD-L1 Expression in Tumor Tissues
2
Confocal Imaging of Cellular Internalization and Endo-lysosomal Escape
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For confocal imaging, 4T1 cells were seeded in Lab-Tek 8-well chamber slides (1 × 104 cells/well) and incubated for 1 d. Then, the cells were incubated with various drugs. Confocal images were taken by the confocal microscope.
To study the cellular internalization of DOX and HDDA, 4T1 cells were treated with DOX or HDDA (DOX: 1.8 μg/mL) for various time periods. Next, the media were replaced by the Hoechst working solution (10 μg/mL) and further incubated for 10 min. Then, the cells were washed with PBS and observed by the confocal microscope at the excitation wavelengths of 405 (for Hoechst) and 552 (for DOX) nm, respectively.
To study the endo-lysosomal escape of HDDA NBs, 4T1 cells were incubated with HDDA NBs (DOX: 3.6 μg/mL) for different time periods. Next, the culture media were replaced by the media containing Hoechst (10 μg/mL) and LysoTracker Green (0.5 μg/mL, purchased from KeyGEN BioTECH, China) and further incubated for 15 min. Finally, the 4T1 cells were washed with PBS and observed by the confocal microscope at the excitation wavelengths of 405 (for Hoechst), 488 (for LysoTracker Green), and 552 (for DOX) nm, respectively.
To study the cellular internalization of DOX and HDDA, 4T1 cells were treated with DOX or HDDA (DOX: 1.8 μg/mL) for various time periods. Next, the media were replaced by the Hoechst working solution (10 μg/mL) and further incubated for 10 min. Then, the cells were washed with PBS and observed by the confocal microscope at the excitation wavelengths of 405 (for Hoechst) and 552 (for DOX) nm, respectively.
To study the endo-lysosomal escape of HDDA NBs, 4T1 cells were incubated with HDDA NBs (DOX: 3.6 μg/mL) for different time periods. Next, the culture media were replaced by the media containing Hoechst (10 μg/mL) and LysoTracker Green (0.5 μg/mL, purchased from KeyGEN BioTECH, China) and further incubated for 15 min. Finally, the 4T1 cells were washed with PBS and observed by the confocal microscope at the excitation wavelengths of 405 (for Hoechst), 488 (for LysoTracker Green), and 552 (for DOX) nm, respectively.
3
Immunocytochemical Staining of NRCM
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NRCM was cultured in a μ-slide 8-well chamber (IBIDI GmbH, Germany) and fixed with freshly prepared 4% paraformaldehyde at room temperature for 15 minutes. Then, NRCM was permeabilized with 0.5% Triton X-100 and 2 mM Ribonucleoside Vanadyl Complexes (Sigma) on ice for 10 min followed by washing twice with 2x SSC for 10 min each time. The probe (10 ng/mL) was denatured in the hybridization buffer at 90°C for 10 minutes and then immediately chilled on ice for 5 min. NRCM was hybridized and incubated with primary antibodies: α-actinin antibody (1 : 200, Sigma) in a hybridization oven at 37°C overnight. After hybridization, the chamber was washed in 2× SSC, 50% formamide for 3 × 5 mins at 42°C, 2× SSC for 3 × 5 mins at 42°C, 1× SSC for 3 × 5 mins at 42°C, 4× SSC for 2 × 10 mins at room temperature, and 2× SSC for 1 hr at 65°C; Cy3-labeled streptavidin and secondary antibodies were added to the NRCM for 1 hr at RT. After washing three times with PBS, Hoechst (1 : 1000, Keygen) was used to label the nucleus for 30 min, and images were captured by a confocal microscope (Carl Zeiss, Thuringia, Germany).
4
Cell Proliferation Assay via EdU Imaging
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EdU Imaging Kit (KeyGEN, Nanjing, China) was used for evaluating cell proliferation according based on the manufacturer's instruction. 5 μg/mL Hoechst (KeyGEN) was used for the staining of cell nuclei. EdU-positive cell number was measured by fluorescence microscope (Olympus, Tokyo, Japan).
5
Immunostaining of Cardiomyocytes and Heart Tissue
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Sections (5 μm) and NRCMs were fixed, permeabilized, and blocked. Then, the sections or NRCMs were incubated with primary antibodies: α-actinin antibody (1 : 200, Sigma), Ki67 antibody (1 : 200, Abcam), and phospho-histone H3 (Ser10) polyclonal antibody (1 : 100, Invitrogen). After incubation of secondary antibodies, Hoechst (1 : 1000, Keygen) was used to label the nucleus for 30 min. The images of NRCMs were collected with a fluorescence microscope (Leica, Wetzlar, Germany), and the images of heart sample sections were collected by a confocal microscope (Carl Zeiss, Thuringia, Germany). All analyses were performed by investigators blinded to the treatment.
6
Immunofluorescence Quantification of CD8+ T Cells
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For the inoculation on the bilateral sides of each female ICR mouse (N = 7), the inoculation was performed by subcutaneous injection of H22 (2 × 105) cell suspension on bilateral sides of each mouse. After 1 week, the primary tumors of mice were treated by PTT via the intravenous injection of PLOV or surgery. The distal tumor tissues were isolated from mice in 5th day or 10th day after treatment. The collected tumors were placed in 4% paraformaldehyde for 6 to 8 h and transferred to 20% sucrose and the frozen tissue pieces were embedded. A frozen tissue section of 8 μm thick was prepared by a cryostat fixed with acetone for 10 min, and stored at − 20℃ for immunofluorescence. The above sections were washed three times with PBS solution for 3 to 5 min each time. Subsequently, the sections were then blocked with 2% BSA at room temperature for 1 h. After incubation with CD8+ primary antibody (eBioscience) overnight at 4 ℃, the slices were washed three times with PBS. Subsequently, it was incubated with a secondary antibody at room temperature for 1 h, and washed with PBS. After staining with Hoechst (keygen) for 10 min, it was washed twice with PBS, an anti-quenching agent was added dropwise to seal, and it observed using a confocal scanning laser microscope (CSLM) (Olympus, FV1000).
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