The sequence containing the human Sirt1 gene was selected using Invitrogen designer software. Small interfering RNAs that can efficiently silence human Sirt1 were validates. Retroviruses were generated by transfecting empty plasmid vectors containing Sirt1, small interfering RNA targeting human Sirt1 and enhanced green fluorescence protein (EGFP) into 293T packaging cells. Finally, four recombinant lentiviral vectors were constructed: Vector1 (lentivirus-EGFP), Sirt1 (lentivirus-Sirt1-EGFP), Si-sirt1 (lentivirus-EGFP-Si-Sirt1), and Vector2 (lentivirus- EGFP-pRNAi). pRNAi was used as the negative control and a scrambled non-targeting sequence. For transfection, LY-3 and LY-10 cells were plated onto 12-well plates at 2.5x105 cells/well and infected with the lentiviral stocks at a multiplicity of infection, in the presence of polybrene (10 μg/ml), and then analyzed using uorescence microscopy (Olympus, Tokyo, Japan) and western blotting at 72 hours post-transduction. Further, each group of EGFP-positive cells was sorted using a flow sorter.
Uorescence microscopy
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Fluorescence microscopy is an imaging technique that uses fluorescent probes to visualize and study biological samples. The core function of this equipment is to excite fluorescent molecules within the sample with a specific wavelength of light, causing them to emit light at a longer wavelength. This allows for the selective observation and analysis of specific structures, proteins, or cellular components within the sample.
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12 protocols using uorescence microscopy
1
Lentiviral-Mediated Sirt1 Regulation
2
Histological Assessment of Spinal Cord Inflammation
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Lumbar spinal cord sections were stained with hematoxylin-eosin (HE) to assess in ammation. First, the sections were degreased with 95% ethanol for 10 min and transferred into hematoxylin solution to stain nuclei for 2 min. The sections were then rinsed in running tap water, immersed in 0.25% ammonia, then redyed with eosin solution for 1.5 min. Finally, the sections were washed in deionized water, dehydrated in alcohol (95%, 100%(I), 100%(II)) for 3 min each, treated with xylene, and sealed with resinene. Images were photographed and analyzed by Olympus Corporation uorescence microscopy.
3
Immunohistochemical Labeling of Microglia and Macrophages
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For immunohistochemistry experiments, spinal cord sections were incubated with primary antibody dissolved in blocking solution (3% BSA and 0.3% Triton X-100 in PBS) overnight at 4°C. Microglia were labelled with IBA-1 Rabbit mAb (1:100, CST) and macrophages with Puri ed Rat CD107b (1:30, BD Pharmingen, USA). Next, sections were washed with PBS and incubated with HRP-linked secondary antibody (1:200) at room temperature for 1 h. After an additional PBS wash, a DAB-peroxidase reaction was carried out for 2-8 min. The sections stained with Puri ed Rat CD107b (1:30, BD Pharmingen, USA) were counterstained with haematoxylin. All the sections were then dehydrated in alcohol (85%, 95%, 100%(I), 100%(II)) each for 10 min, treated with xylene, and sealed with resinene. Images were photographed and analyzed by Olympus Corporation uorescence microscopy.
4
Quantifying Cellular ROS Levels
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ROS assay kit (Solarbio,China) with 2,7-dichlorodihydro uorescein diacetate (DCFH-DA) helped to determine the ROS levels in these cells. RAW264.7 were seeded on a 6-well plate at a density of 2 × 10 5 cells each well and treated by using various regents. After 72 h treatments, the cells received 20 min of incubation by using 10 µM DCFH-DA in cell incubator at 37℃ and then washed by using serum-free DMEM media three times. The uorescence microscopy (OLYMPUS Tokyo, Japan) was used to obtain the images.
5
Adhesion of THP-1 Cells to HUVECs
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After HUVECs were seeded in a 6-well cell culture plate for 24 h, they were treated with 7-Mif (50 µM) or vehicle (DMSO) for 15 minutes, followed by IL-1β (10 ng/mL) or PBS for 4 hours. Labeling with 5 µM Vybrant DiD was performed for 30 minutes according to manufactures' instruction. Labeled THP-1 cells were then added to medium containing HUVECs and incubated for 30 min. THP-1 cells that didn't bound to HUVECs were removed by washing with PBS. The amount of adhering green uorescent THP-1 cells were measured with uorescence microscopy (Olympus, USA).
6
Cellular Morphology Changes Assessed by Hoechst Staining
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The changes in cellular morphology of HTB9 and BIU87 cells were observed by using Hoechst 33258 staining (Sigma) as previously described 25 . Brie y, HTB9 and BIU87 cells treated with or without 3.82mg/ml or 2.85 MI were seeded into 6-well plates and cultured overnight. Then cells were xed with 4% formaldehyde for 15 min, and stained in Hoechst 33258 (10mg/ L) for another 1 h. After washing with PBS for twice, cells were subjected to uorescence microscopy (Olympus, Tokyo, Japan). Meanwhile, the morphological changes including reduction in the volume and nuclear chromatin condensation were observed.
7
Tracking BM-MSCs in Tumor Microenvironment
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BM-MSCs were transduced with a lentiviral vector expressing the enhanced green uorescent protein (eGFP) gene (a gift from Stem Cell Technology Research Center, Tehran, Iran) at a multiplicity of infection of 10 (MOI=10) and the transduction e ciency was evaluated directly in cell culture using uorescence microscopy (Olympus, Tokyo, Japan) after 24h (Fig 2).
In order to track the migration and distribution of injected BM-MSCs transduced with eGFP (MSC-eGFP) in the tumor microenvironment, 1.5×10 6 MSCs at the third passage in 100 μL of PBS were injected into tumor-bearing mice through the peritumoral administration procedure. Mice (n=3/group) were sacri ced under deep anesthesia after the injection, and the intensity of uorescent signal was evaluated in tumor sections.
In order to track the migration and distribution of injected BM-MSCs transduced with eGFP (MSC-eGFP) in the tumor microenvironment, 1.5×10 6 MSCs at the third passage in 100 μL of PBS were injected into tumor-bearing mice through the peritumoral administration procedure. Mice (n=3/group) were sacri ced under deep anesthesia after the injection, and the intensity of uorescent signal was evaluated in tumor sections.
8
Neuronal Apoptosis Detection Protocol
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After anesthesia, the rats were perfused transcardially with 4℃ PBS followed by 4% paraformaldehyde. Then, the brain tissue was taken out immediately and xed in 4% paraformaldehyde overnight. Sucrose solution was dehydrated in gradient, after rinsed, the brain tissue was quickly frozen on the machine.
Finally, the continuous coronal frozen section was made. TUNEL and staining for NeuN, a neuronal marker, were used together to detect neuronal apoptosis. Brie y, frozen sections were rewarmed at room temperature for 20 min, blocked with 5% sheep serum(Equitech-Bio,SS-0100) for 1 h and then incubated with anti-NeuN primary antibody (Cell Signaling Technology, cat# 12943, 1:200 dilution). A TUNEL kit (Roche, cat# 11684795910) was used to label apoptotic cells after the use of anti-NeuN secondary antibody (Alexa Fluor® Plus 594-conjugated). The sections were incubated with 50 μL of TUNEL reaction mixture (enzyme solution:labeling solution = 1:9), incubated at 37°C in the dark for 60 min, and washed 3 times with PBS for 5 min each, then the sections were sealed and observed by uorescence microscopy (Olympus, Tokyo, Japan). TUNEL-positive cells in ve different elds were counted. The results are expressed as cells/mm2, and the apoptotic ratio was calculated as the number of apoptotic cells/the total cell number × 100%.
Finally, the continuous coronal frozen section was made. TUNEL and staining for NeuN, a neuronal marker, were used together to detect neuronal apoptosis. Brie y, frozen sections were rewarmed at room temperature for 20 min, blocked with 5% sheep serum(Equitech-Bio,SS-0100) for 1 h and then incubated with anti-NeuN primary antibody (Cell Signaling Technology, cat# 12943, 1:200 dilution). A TUNEL kit (Roche, cat# 11684795910) was used to label apoptotic cells after the use of anti-NeuN secondary antibody (Alexa Fluor® Plus 594-conjugated). The sections were incubated with 50 μL of TUNEL reaction mixture (enzyme solution:labeling solution = 1:9), incubated at 37°C in the dark for 60 min, and washed 3 times with PBS for 5 min each, then the sections were sealed and observed by uorescence microscopy (Olympus, Tokyo, Japan). TUNEL-positive cells in ve different elds were counted. The results are expressed as cells/mm2, and the apoptotic ratio was calculated as the number of apoptotic cells/the total cell number × 100%.
9
Tracking BM-MSCs Migration in Tumors
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BM-MSCs were transduced with a lentiviral vector expressing the enhanced green uorescent protein (eGFP) gene (a gift from Stem Cell Technology Research Center, Tehran, Iran) at a multiplicity of infection of 10 (MOI=10) and the transduction e ciency was evaluated directly in cell culture using uorescence microscopy (Olympus, Tokyo, Japan) after 24h (Fig 2 ).
In order to track the migration and distribution of injected BM-MSCs transduced with eGFP (MSC-eGFP) in the tumor microenvironment, 10 5 MSCs at the third passage in 100 μL of PBS were injected into tumorbearing mice through the peritumoral administration procedure. Mice (n=3/group) were sacri ced under deep anesthesia after the injection, and the intensity of uorescent signal was evaluated in tumor sections.
In order to track the migration and distribution of injected BM-MSCs transduced with eGFP (MSC-eGFP) in the tumor microenvironment, 10 5 MSCs at the third passage in 100 μL of PBS were injected into tumorbearing mice through the peritumoral administration procedure. Mice (n=3/group) were sacri ced under deep anesthesia after the injection, and the intensity of uorescent signal was evaluated in tumor sections.
10
Spinal Cord Injury Impacts MMP-7 Expression
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The cellular distribution of MMP-7 expression was measured by immuno uorescence staining at the 24h time point after SCII. Spinal cord segments were xed and cut into 8 µm sections. The segments were sealed with 10% bovine serum albumin (BSA) and incubated with primary antibodies overnight at 4°C. Then, they were incubated with the corresponding secondary antibodies for 2 h at room temperature.
Primary antibodies included mouse anti-NeuN (Abcam, ab104224, 1:400), goat anti-GFAP (Abcam, ab53554, 1:400), mouse anti-Iba-1 (Abcam, ab5076, 1:400), rabbit anti-MMP-7 (A nity, AF0218, 1:200) and mouse anti-cleaved caspase-3 (Abmart, M020539, 1:50). Corresponding secondary antibodies included FITC-conjugated A niPure donkey anti-goat IgG (H+L) (Proteintech, 1:200), Alexa Fluor 594conjugated donkey anti-rabbit IgG (H+L) (Proteintech, 1:200), uorescein (FITC)-conjugated A niPure donkey anti-mouse IgG (H+L) (Proteintech, 1:200). Images of anterior horns were captured under a uorescence microscopy (Olympus, Melville, USA).
Primary antibodies included mouse anti-NeuN (Abcam, ab104224, 1:400), goat anti-GFAP (Abcam, ab53554, 1:400), mouse anti-Iba-1 (Abcam, ab5076, 1:400), rabbit anti-MMP-7 (A nity, AF0218, 1:200) and mouse anti-cleaved caspase-3 (Abmart, M020539, 1:50). Corresponding secondary antibodies included FITC-conjugated A niPure donkey anti-goat IgG (H+L) (Proteintech, 1:200), Alexa Fluor 594conjugated donkey anti-rabbit IgG (H+L) (Proteintech, 1:200), uorescein (FITC)-conjugated A niPure donkey anti-mouse IgG (H+L) (Proteintech, 1:200). Images of anterior horns were captured under a uorescence microscopy (Olympus, Melville, USA).
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