The JC-1 assay kit (Beyotime, China) is universally used to detect the mitochondrial membrane potential. JC-1 aggregated in the mitochondrial matrix at high membrane potential and could form red fluorescent aggregates. However, JC-1 cannot aggregate to form a polymer at low membrane potential and produce green fluorescent monomers. Briefly, cells (1 × 104) were incubated into 24-well plates for 5 days after drug treatment. The cells were The JC-1 staining solution was added for 20 min, incubated with JC-1 dye at 37 °C for 20 min, washed twice with dilution buffer, and placed in the culture medium. and the fluorescence intensity was obtained by laser scanning confocal microscopy (40×, Leica, Germany) Lastly, images were obtained by laser scanning confocal microscopy (40×, Leica, Germany) at 490 nm excitation and 530 nm emission for green and at 525 nm excitation and 590 nm emission for red. Mitochondrial membrane potential is expressed as the ratio of red fluorescence (JC-1 aggregates) to green fluorescence (JC-1 monomers) using the ImageJ software.
Confocal laser scanning microscopy
Manufactured by Leica
Sourced in Germany, United States, Japan
Confocal laser scanning microscopy is an optical imaging technique that uses a focused laser beam to scan samples point-by-point. It captures high-resolution, three-dimensional images by rejecting out-of-focus light, resulting in improved contrast and resolution compared to conventional optical microscopy.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Beyotime (10 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (6 mentions)
Triton x 100, by Merck Group (5 mentions)
Ix70 inverted microscope, by Olympus (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Live dead baclight bacterial viability kit, by Thermo Fisher Scientific (4 mentions)
Nicolet 6700, by Thermo Fisher Scientific (4 mentions)
Normal goat serum (ngs), by Zhongshan Golden Bridge Biotechnology (3 mentions)
Alexa fluor 488 goat anti rabbit igg, by Abcam (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (3 mentions)
Mitotracker red, by Thermo Fisher Scientific (3 mentions)
Confocal laser scanning microscope, by Leica (3 mentions)
Alexa fluor 488, by Abcam (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (3 mentions)
Alexa fluor 647 dextran conjugate, by Thermo Fisher Scientific (3 mentions)
Fluoview fvx confocal scan head, by Olympus (2 mentions)
C1002, by Beyotime (2 mentions)
Glial fibrillary acidic protein (gfap), by Zhongshan Golden Bridge Biotechnology (2 mentions)
Bovine serum albumin (bsa), by Beyotime (2 mentions)
253 protocols using confocal laser scanning microscopy
1
Quantifying Mitochondrial Membrane Potential
2
TGF-β Immunofluorescence Staining Protocol
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Cells were washed with PBS and fixed in 4% paraformaldehyde at 4°C for 15 min at room temperature. Cells were blocked with 5% BSA and 0.25 Triton X-100 for 1 h. Cells were incubated with TGF-β at 4°C overnight. Cells were washed with PBST, 555-secondary antibodies (HRP conjugated, PerkinElmer, Inc., Waltham, MA, USA) were used for 1 h at 37°C. Cells were stained with DAPI assay for 15 min at darkness and washed with PBST for 15 min. Laser scanning confocal microscopy (Leica Microsystems GmbH, Wetzlar, Germany) was used for cell observation.
3
Immunofluorescence Staining of H. pylori
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For human gastric epithelial cells, cells were firstly fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 solution. For mouse stomach sections, slides were firstly deparaffinized and rehydrated, and permeabilized with 0.5% Triton X-100 solution. Then, 10% goat serum was used to block non-specific antigens in both cells and mouse stomach sections. Slides were incubated with primary antibody (anti-H. pylori (Abcam, ab7788, 1:200), anti-H. pylori (Abcam, ab231433, 1:200), anti-LOX-1 (Abcam, ab60178, 1:100), anti-His-Tag (Santa Cruz Biotechnology, sc-8036, 1:50), anti-Lewis b (Santa Cruz Biotechnology, sc-51513, 1:50)) 4 °C overnight followed by incubation of secondary antibody (Goat anti-rabbit conjugated to Alexa Fluor 488 (Invitrogen, A-11008, 1:500), goat anti-rabbit conjugated to Alexa Fluor 568 (Invitrogen, A-11011, 1:500), goat anti-mouse conjugated to Alexa Fluor 568 (Invitrogen, A-11031, 1:500)) and then 4’,6-diamidino-2-phenylindole (DAPI) to stain the nucleus. Sections were evaluated using laser scanning confocal microscopy (Leica Microsystems, Wetzlar, Germany).
4
In vivo pEGFP Transfection of Mice
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Forty-two male KM mice (weighing 20–30 g each) at 4~5 weeks of age were randomly divided into three groups for in vivo transfection of pEGFP-loaded TACS and TACS-HBC composite particles. Group A was transfected with pEGFP-loaded TACS core particles. Group B was transfected with pEGFP-loaded TACS-HBC composite particles, and Group C, the control group, was transfected with saline. The mice were first anaesthetized by intraperitoneal injection with 3% chloral hydrate according to a standard of 0.01 mL/g/mouse to make the skeletal muscles loosen and to prevent pEGFP from being extruded by muscle contractions. Both hind legs of each mouse were sterilized with alcohol after the hair was shaved, and 50 μL of 25 g/L hypertonic sucrose (China Otsuka Pharmaceutical Co., Ltd., Tianjing, People’s Republic of China) was injected into the quadriceps muscle. Fifteen minutes post-injection, the solutions containing pEGFP-loaded particles (20 μg pEGFP) were injected into the quadriceps slowly. The needle remained in the muscle 5~10 seconds, and was then removed slowly to prevent injected solutions from exuding. pEGFP expression was detected by laser scanning confocal microscopy (Leica Microsystems, Wetzlar, Germany) after quadriceps muscle sections were cut and frozen.
5
Multicolor Immunofluorescence Imaging of Neuronal and Glial Markers
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Tissue sections (10 μm) were incubated in normal goat serum (Zhongshan Golden Bridge, Beijing, China) for 30 min followed by incubation with a mixture containing a PDE10A antibody (rabbit polyclonal antibody, 1:100), a microtubule-associated protein 2 (MAP2) antibody (chicken polyclonal antibody, 1:100, Zhongshan Golden Bridge) and a glial fibrillary acidic protein (GFAP) antibody (mouse polyclonal antibody, 1:100, Zhongshan Golden Bridge) overnight at 4°C. Sections were washed twice in PBS and incubated with a mixture of DyLight 488-conjugated goat anti-rabbit IgG (1:200, Zhongshan Golden Bridge), DyLight 549-conjugated goat anti-mouse IgG (1:200, Zhongshan Golden Bridge) and DyLight 405-conjugated goat anti-chicken IgG (1:200, Zhongshan Golden Bridge) in a darkroom for 90 min at 37°C. Tissue sections were mounted in 50% glycerol/PBS. Fluorescence was detected using laser scanning confocal microscopy (Leica Microsystems Heidelberg GmbH, Germany) on an Olympus IX 70 inverted microscope (Olympus, Japan) equipped with a Fluoview FVX confocal scan head. To determine the specificity of antibodies, PDE10A antibody (488 channel), GFAP antibody (549 channel) and microtubule-associated protein 2 (MAP2) antibody (405 channel) were replaced by PBS with the same protocols.
6
Quantifying DNA Double-Strand Breaks
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Cells were exposed to 6.0 Gy X‐radiation and recovered for 48 hours. The cells were grown on the tunnel of μ‐slide VI (Ibidi). Cells were fixed by polyformaldehyde (4%) for 30 minutes, followed by treatment with 0.2% Triton X‐100 for 10 minutes. The antibody to γH2AX (1:100, Abcam; #ab2893) was diluted using 3% bovine serum albumin and incubated with the permeabilized cells. Following wash procedure using phosphate buffer saline (PBS), the secondary antibody Alexa Fluor 488 goat anti‐rabbit antibody to immunoglobulin G (1:2000, Cell Signaling Technology; #4412S) was then added and incubated for 1 hour. Nucleus was stained using 4′,6‐diamidino‐2‐phenylindole (DAPI), and immunofluorescence images were obtained using laser‐scanning confocal microscopy (Leica Microsystems).
7
Immunostaining Optimization for IHC and IF
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Epitope retrieval for IHC and IF staining were performed as previously described [16 (link)]. Primary antibodies and dilution for IHC and IF staining are shown in Supplementary Table 2 . HRP-goat anti-mouse IgG conjugate (Cell Signaling Technology) and HRP-goat anti-rabbit IgG conjugate (Life Technologies) were used as secondary antibodies. Chromogenic color development was performed using 3,3’-diaminobenzidine enhanced liquid substrate (Sigma, St. Louis, MO, USA) and nuclei counterstained by Mayer's hematoxylin (Sigma). IF staining for YAMC cells and mouse colon biopsies was performed as previously described [34 (link)]. Goat anti-rabbit IgG (H+L)-Alexa 647 conjugate and goat anti-mouse IgG (H+L)-Alexa 647 conjugate (Life Technologies) were used as secondary antibodies. Nuclei were counterstained by 4’,6-diamidino-2-phenylindole. Images were analyzed by laser scanning confocal microscopy (Leica Microsystems, Buffalo Grove, IL, USA).
8
Mitochondrial Imaging via MitoTracker
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Following washing with pre-warmed PBS three times, the cells were incubated in the dark with 100 nmol/l MitoTracker Red working solution (Thermo Fisher Scientific Inc.) at 37°C for 15 min. Following incubation, the cells were washed three times and then directly visualized by laser scanning confocal microscopy (magnification, ×630; Leica Microsystems GmbH).
9
Immunofluorescence Staining of YAMC and Tissue
10
Measuring Cell Proliferation via EdU Labeling
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Cells were transfected with siRNAs (Mock-siRNA, siL21-1 and siL21-2) at a concentration of 40 nM for 72 h. The transfected cells were exposed to 50 μM of 5-ethynyl-2′-deoxyuridine (EdU) (RiboBio, Guangzhou, China) for 2 h at 37°C, and the cells were fixed in 4% paraformaldehyde for 30 min at room temperature, followed by the addition of 2 mg/ml glycine to neutralize the reaction. After permeabilization with 0.5% Triton-X, the cells were reacted with Apollo reaction cocktail (RiboBio, Guangzhou, China) for 30 min in the dark at room temperature. Subsequently, the DNA contents of the cells were stained with Hoechst 33342 for 30 min and visualized under a laser scanning confocal microscopy (Leica Microsystems, Buffalo Grove, IL, United States).
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