Mice were fasted for 6 hours before sacrifice. Tissue samples were dissected, fixed in 4% paraformaldehyde for 48 hours, and embedded in paraffin blocks. H&E stains were performed by the Histology Core in the Department of Comparative Medicine. For immunofluorescent staining, mice were anesthetized for intracardial perfusion of PBS, followed by 4% paraformaldehyde with 0.08% glutaraldehyde. Brain and adipose depots were dissected and postfixed in 4% paraformaldehyde overnight. Coronal brain sections (50 μm) were prepared using a vibratome. Tissue sections were blocked with 3% BSA and 0.2% Triton X-100 in 0.1 M phosphate-buffered saline (PBS) and were incubated with primary antibodies (1:100 dilution; CST, anti-OGT, 24083; Abcam, anti-RL2, ab2739) overnight and secondary antibodies [1:400; Alexa Fluor 488 anti-rabbit immunoglobulin G (IgG), Alexa Fluor 594 anti-rabbit IgG, Alexa Fluor 594 anti-mouse IgG, and Alexa Fluor 647 anti-rabbit IgG] for 1 hour. Immunofluorescent staining and clearance of tissues were performed following previous literature (72 ). The slides were mounted with VECTASHIELD antifade mounting medium (Vector Laboratories) and saved at 4°C until imaging. A Keyence digital microscope and the Leica SP5 confocal system were used for H&E stain imaging and fluorescent imaging.
Digital microscope
Manufactured by Leica camera
Sourced in United States
The Digital Microscope is a precision imaging device that captures high-resolution digital images of microscopic subjects. It features a high-quality lens system and advanced image sensor to provide detailed, clear visualizations of small-scale specimens.
Automatically generated - may contain errors
Lab products found in correlation
Sp5 confocal system, by Leica camera (1 mentions)
Vectashield antifade mounting medium, by Vector Laboratories (1 mentions)
Plcg1, by Cell Signaling Technology (1 mentions)
Bioluminescence imaging, by PerkinElmer (1 mentions)
Anti rabbit, by Cell Signaling Technology (1 mentions)
Matrigel, by Corning (1 mentions)
Matrigel, by BD (1 mentions)
Anti sqstm1 p62, by Cell Signaling Technology (1 mentions)
Interleukin 6 (il 6), by Wuhan Servicebio Technology (1 mentions)
Masson s trichrome staining kit, by Wuhan Servicebio Technology (1 mentions)
Anti cd31, by Abcam (1 mentions)
11 protocols using digital microscope
1
Immunofluorescent Analysis of OGT and RL2 in Mouse Tissues
2
In Vivo Xenograft Tumor Model
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Packaged luciferase lentiviruses were purchased from Shanghai Bioegene Co., Ltd. Infected cells were treated with puromycin for 3 days to select a stable cell line.
Four-week-old male NOD-SCID mice with body weights ranging from 23 to 26 g were subcutaneously injected with 100 μl of Matrigel (Corning) containing 8 × 106 tumor cells. Tumor sizes and the weights of the mice were measured every other day. The size of xenografts was evaluated with bioluminescence imaging (PerkinElmer, Waltham, Massachusetts, USA). Mice were euthanized 20 days after SW1088 cell implantation. All animal experiments were approved by the Institutional Animal Care and Use Committee of Fudan University.
The tumors from xenografts were collected and fixed with 4% paraformaldehyde solution and embedded in paraffin. The sliced tissues (thickness: 4 mm) mounted on the glass slides were incubation by primary antibody (PLCG1, Cell Signaling Technology) and secondary antibody (Anti-rabbit, Cell Signaling Technology), then via DAB chromogenic reaction and visualized using a digital microscope (Leica). The cell nuclei were stained with DAPI. Briefly, to score the IHC staining data, five images of each sample were taken and the average integrated optical density was measured by Image Pro Plus 6.0 software.
Four-week-old male NOD-SCID mice with body weights ranging from 23 to 26 g were subcutaneously injected with 100 μl of Matrigel (Corning) containing 8 × 106 tumor cells. Tumor sizes and the weights of the mice were measured every other day. The size of xenografts was evaluated with bioluminescence imaging (PerkinElmer, Waltham, Massachusetts, USA). Mice were euthanized 20 days after SW1088 cell implantation. All animal experiments were approved by the Institutional Animal Care and Use Committee of Fudan University.
The tumors from xenografts were collected and fixed with 4% paraformaldehyde solution and embedded in paraffin. The sliced tissues (thickness: 4 mm) mounted on the glass slides were incubation by primary antibody (PLCG1, Cell Signaling Technology) and secondary antibody (Anti-rabbit, Cell Signaling Technology), then via DAB chromogenic reaction and visualized using a digital microscope (Leica). The cell nuclei were stained with DAPI. Briefly, to score the IHC staining data, five images of each sample were taken and the average integrated optical density was measured by Image Pro Plus 6.0 software.
3
Organ Histology Analysis in Mice
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Mice in two groups (PBS and SOR@LDH-CuS/P) were sacrificed after 16 days of treatment. Then, heart, liver, spleen, lungs and kidneys were collected and preserved in 4% paraformaldehyde solution for histology analysis. All tissue slices were observed with a Leica digital microscope after stained with H&E.
4
Transwell Invasion and Migration Assays
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The invasion and migration assays were performed using Transwell chambers with 8 μm core filters (Corning, #3422). There was pre-diluted Matrigel (diluted at a ratio of 1:3 with serum-free DMEM medium) (BD, #356234) in filters for invasion assays, while no Matrigel was used for the migration assays. Briefly, 2×104 cells/100 μl with serum-free DMEM was added to the upper chamber, while 600 μl complete medium was added to the lower chamber. After 36 h, cells on the bottom surface were fixed with 4% paraformaldehyde for 15 min, washed by PBS, stained by 0.1% crystal violet at 37 °C for 30 min and observed under a digital microscope (Leica).
5
Stomatal Aperture Analysis in Arabidopsis
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Four-week-old WT and GmbZIP19-OE Arabidopsis were used to observe and measure the stomatal aperture conditions. Leaves of similar size, age and side branches of stems were selected to compare stomatal aperture. At first, the leaves and stems were infused in stomatal solution containing: 50 mM CaCl2, 10 mM MES, 5 mM KCl, pH 6.15 and exposed to light for approximately 2 h to ensure all the stomatal had opened. Subsequently, 150 mM NaCl and 300 mM mannitol were added to the solutions to induce salt and drought stress conditions, respectively. After 2 h of treatments, leaves and stems of WT and GmbZIP19-OE Arabidopsis were blended and observed with a digital microscope (Leica). The length and width of stomata were measured using image analysis (Adobe photoshop CC 2019) computer software [55 (link)].
To confirm the phenotype and quantitative result, we examined the expression levels of marker genes of stomatal movement and closure using GsmbZIP19-OE plants under salt or drought treatment. The stomatal movement marker genes are: WRKY54 and WRKY70, the primers are listed inTable S2 .
To confirm the phenotype and quantitative result, we examined the expression levels of marker genes of stomatal movement and closure using GsmbZIP19-OE plants under salt or drought treatment. The stomatal movement marker genes are: WRKY54 and WRKY70, the primers are listed in
6
Nrf2 and p62 Immunofluorescence Imaging
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ACC-83 cells were seeded to a 24-well plate with the coverslip, being treated with proteasome inhibitor MG132 for 24 h when the cell reached a confluence of 60 ~ 70% per plate. The cells were washed twice by PBS and fixed with 4% paraformaldehyde solution for 1 h in the indoor temperature, following being rinsed thrice by PBS. After that, the cells were permeabilized and blocked with PBS which contained 0.5% Triton X-100 and being blocked with 1% bovine serum albumin PBS for 20 min at RT. Rinsed thrice by PBS the same as the former. Cells were incubated with primary antibody against Nrf2 (CST, Cell Signaling Technology, Rabbit)and Anti-SQSTM1/p62 (CST, Cell Signaling Technology, Rabbit) both at a dilution of 1:100 at 4 °C overnight. After being washed with PBS thrice, the 488(Abcam, Goat Anti-Rabbit)and 594(Abcam, Goat Anti-Rabbit)secondary antibodies were incubated in the same conditions with the primary antibody for 1 h at 37℃. Cell washed thrice with PBS. Following DAPI (10 µg/ml) solution was added. Before imaging cells were rinsed thrice for 5 min per wash. Fluorescence images were captured by a Leica digital microscope. Images were merged using Adobe Photoshop CS6 without any modifications.
7
Tissue Fixation, Embedding, and Imaging
8
Quantifying Tumor Angiogenesis in Mice
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We also investigated the variation of blood vessels of mice after different treatments. FITC-labeled CD34 and Cy 3-labeled CD31 immunofluorescence staining assays were performed on tumor slices. The corresponding fluorescence images were then captured by a Leica digital microscope.
9
Tumor Histopathology After Treatment
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All the mice in six groups were sacrificed after 16 days of treatment. Then, the tumors and major organs of each group were collected and preserved in 4% paraformaldehyde solution for histology analysis. All tissue slices were observed with a Leica digital microscope after stained with H&E and TUNEL.
10
Calcein-AM/PI Assay for Therapeutic Evaluation
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Calcein-AM/PI assay was utilized to visualize the therapeutic effect. Briefly, HepG2 cells (1 × 105 cells/well) were inoculated into 6-well plates for 24 h incubation at 37 °C with 5% CO2, followed by treatment with 100 µL of PBS, NIR (0.5 W cm− 2, 6 min), LDH-CuS/P (concentration of LDH-CuS: 100 µg mL− 1), LDH-CuS/P + NIR (concentration of LDH-CuS: 100 µg mL− 1, 0.5 W cm− 2, 6 min), SOR@LDH-CuS/P (concentration of LDH-CuS: 100 µg mL− 1, LDH-CuS: SOR = 1: 2) and SOR@LDH-CuS/P + NIR (concentration of LDH-CuS: 100 µg mL− 1, LDH-CuS: SOR = 1: 2, 0.5 W cm− 2, 6 min) for 24 h, respectively. The NIR irradiation was performed at 12 h post-administration and divided into 3 times, each of two-minutes duration with an interval of 10 min. Subsequently, fresh DMEM containing Calcein-AM (2 mL, 5 µg mL− 1) and PI (2 mL, 10 µg mL− 1) were added into 6-well plates for 30 min incubation. All treated cells were washed with PBS three times and the corresponding fluorescence images were captured by a digital microscope (Leica).
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