The CKX 41 inverted fluorescence microscope was purchased from Olympus (Tokyo, Japan) and the mini electrophoresis meter and microplate reader were purchased from Bio-Rad (Hercules, CA, USA). The EPICS XL flow cytometer was purchased from Beckman Coulter (Brea, CA, USA); the SP2 laser confocal scanning microscope was purchased from Leica (Solms, Germany) and the CO-150 CO2 incubator was purchased from New Brunswick Scientific (Edison, NJ, USA).
Ckx41 inverted fluorescence microscope
Manufactured by Olympus
Sourced in Japan
The CKX41 is an inverted fluorescence microscope designed for laboratory use. It provides basic functionality for visualizing fluorescently-labeled samples. The microscope features an LED illumination system and filters for common fluorescent dyes. Its core function is to allow users to observe and capture images of fluorescent specimens.
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Lab products found in correlation
Co 150 co2 incubator, by Eppendorf (2 mentions)
Epics xl flow cytometer, by Beckman Coulter (2 mentions)
Microplate reader, by Bio-Rad (1 mentions)
8 chamber slide, by BD (1 mentions)
Tetrabutylammonium hydroxide, by Merck Group (1 mentions)
2 isocyanatoethyl methacrylate, by Merck Group (1 mentions)
Dta 50, by Shimadzu (1 mentions)
β cyclodextrin, by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Tga 50, by Shimadzu (1 mentions)
Poly ethylene glycol 600 peg 600, by Merck Group (1 mentions)
Cc12 fluorescence camera, by Olympus (1 mentions)
Hydrogen peroxide, by Beyotime (1 mentions)
Paraformaldehyde (pfa), by Beyotime (1 mentions)
Cellp imaging software, by Olympus (1 mentions)
Goat anti rabbit, by Solarbio (1 mentions)
Gel doc xr imaging system, by Bio-Rad (1 mentions)
Model 680 microplate reader, by Bio-Rad (1 mentions)
9 protocols using ckx41 inverted fluorescence microscope
1
Microscopy and Flow Cytometry Equipment
2
NF-κB Activation in RAW264.7 Cells
3
Comprehensive Characterization of Polymer-Based Biomaterials
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Chemical
and physical characterizations were performed using Shimadzu TGA-50,
Shimadzu DTA-50, Shimadzu DSC-60, Park Systems XE-100E AFM, LEO-EVO
40× SEM, Bruker NMR 300 MHz Ultrashield, Ascend 600 ULW, and
PerkinElmer UATR Two FTIR devices. All spectrophotometric analyses
were carried out with a Shimadzu UV-1601 UV–visible spectrophotometer
and BioTek Eon Elisa microplate reader. The adhesion results were
obtained with an MTS E42 test analyzer. L-929 cell morphologies were
determined by a JuliFL cell analyzer and an Olympus CKX41 inverted/fluorescence
microscope. Histological analysis was carried out with Leica DFC-280
microscope and Leica Q Win Image Analyze System (Leica Micros Imaging
Solutions Ltd., Cambridge, UK).
Polyethylene glycol 200 (PEG200),
polyethylene glycol 400 (PEG400), and polyethylene glycol 600 (PEG600)
were purchased from Merck. 4,4′-Methylenebis cyclohexyl diisocyanate,
β-cyclodextrin, silk sericin (10–40 kDa), toluene-4-sulfonyl
isocyanate, tetrabutylammonium hydroxide, MTT (thiazolyl blue tetrazolium
bromide), Dulbecco’s modified Eagle’s medium (DMEM),
gentamicin sulfate, and 2-isocyanatoethyl methacrylate were purchased
from Sigma-Aldrich. DMSO, acetonitrile, methanol, ethanol, and other
solvents were obtained from Merck. Irgacure-2959 was purchased from
TCI (Tokyo Chemical Industry). All chemicals were used without further
purification.
and physical characterizations were performed using Shimadzu TGA-50,
Shimadzu DTA-50, Shimadzu DSC-60, Park Systems XE-100E AFM, LEO-EVO
40× SEM, Bruker NMR 300 MHz Ultrashield, Ascend 600 ULW, and
PerkinElmer UATR Two FTIR devices. All spectrophotometric analyses
were carried out with a Shimadzu UV-1601 UV–visible spectrophotometer
and BioTek Eon Elisa microplate reader. The adhesion results were
obtained with an MTS E42 test analyzer. L-929 cell morphologies were
determined by a JuliFL cell analyzer and an Olympus CKX41 inverted/fluorescence
microscope. Histological analysis was carried out with Leica DFC-280
microscope and Leica Q Win Image Analyze System (Leica Micros Imaging
Solutions Ltd., Cambridge, UK).
Polyethylene glycol 200 (PEG200),
polyethylene glycol 400 (PEG400), and polyethylene glycol 600 (PEG600)
were purchased from Merck. 4,4′-Methylenebis cyclohexyl diisocyanate,
β-cyclodextrin, silk sericin (10–40 kDa), toluene-4-sulfonyl
isocyanate, tetrabutylammonium hydroxide, MTT (thiazolyl blue tetrazolium
bromide), Dulbecco’s modified Eagle’s medium (DMEM),
gentamicin sulfate, and 2-isocyanatoethyl methacrylate were purchased
from Sigma-Aldrich. DMSO, acetonitrile, methanol, ethanol, and other
solvents were obtained from Merck. Irgacure-2959 was purchased from
TCI (Tokyo Chemical Industry). All chemicals were used without further
purification.
4
Dual Fluorescent Staining for Apoptosis
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The dual acridine orange/ethidium bromide (AO/EB) fluorescent staining can be employed to distinguish apoptotic related changes to the cell membrane during the progression of apoptosis [90 (link),91 (link)]. Cells were seeded as in 4.9 and incubated overnight at 37 °C. The medium was then replenished, and the DOX-loaded MNPs were added using the half-maximal inhibitory concentrations (IC50) of the drug-loaded MNPs (Table 5 ) obtained from the MTT assay. The cells were incubated overnight at 37 °C, followed by removing the medium and washing the cells with 100 µL of PBS. After that, 10 µL of AO/EB dye was added, and the cells were stained for 5 min. The dye was then removed, and the cells were washed with PBS (100 µL), and viewed under an Olympus CKX41 inverted fluorescence microscope at 100X magnification, and images captured using a CC12 fluorescence camera (Olympus Co., Tokyo, Japan). Apoptotic indices were evaluated using Equation (4):
5
Immunofluorescence Staining of Stress Markers
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All cells were cultured onto slides and fixed with 4% paraformaldehyde (Beyotime) at room temperature for 10 min. Cells were washed with PBS and endogenous peroxidase inactivated using 3% hydrogen peroxide (Beyotime) at 25°C for 5 min. Cells were subsequently blocked using 5% bovine serum albumin solution (BSA; Beyotime) at 25°C for 15 min, then incubated with rabbit anti-human GRP78 monoclonal antibody (1 : 3000, Cat. No. ab108615; Abcam, Cambridge, USA), rabbit anti-human C/EBP-homologous protein (CHOP) polyclonal antibody (1 : 3000, Cat. No. MBS000292; MyBioSource, Vancouver, Canada), rabbit anti-human phosphorylated C-Jun N-terminal kinase (p-JNK) monoclonal antibody (1 : 3000, Cat. No. ab124956; Abcam), and rabbit anti-human caspase-12 polyclonal antibody (1 : 2000, Cat. No. ab62484; Abcam) overnight at 4°C. Subsequently, cells were washed using PBS and incubated using Biotin-labeled goat anti-rabbit antibody (1 : 1000, Cat. No. ab6720; Abcam) for 60 min at 37°C. Finally, cells were observed and images captured using a professional CKX41 inverted fluorescence microscope (Olympus Corp., Tokyo, Japan).
6
Bacterial Viability Assay using SYTO-9 and PI
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The staining procedure was performed with solutions of SYTO-9 and propidium iodide (PI), to differentiate the live bacteria from those with membrane damage. Bacterial suspensions were prepared following manufacturing instructions of commercial BacLight bacterial viability kit L7007 obtained from Molecular Probes, Thermo Scientific (USA). [21] (link). Briefly, an overnight culture of S. aureus was inoculated in fresh MH media and incubated at 37 °C under agitation until reaching an OD600 of 0.3. Afterward, bacteria were incubated for 1 and 3 h with S. areira EO with a final concentration of 2 x MIC (128 µg/mL) or MBC (256 µg/mL). To remove the traces of the growth medium, cell suspensions were washed three times and J o u r n a l P r e -p r o o f resuspended in PBS (10 mM, pH=7.40). Finally, both probes were added to achieve a final concentration of 6 µM of SYTO 9 and 30 µM of PI and incubated for 15 min with gentle agitation protected from light. Samples were imaged in an Olympus CKX 41 inverted fluorescence microscope coupled with an Olympus QColor3-RTV-R digital camera (Tokyo, Japan). The proportion of live and dead cells was determined by counting representative images taken from each condition repeated in triplicate using different batches of bacteria.
7
Microglia and Neutrophil Activation in ICH
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We first divided the mice into an ICH group and a PBS group and observed iba1 antibody cells and MPO staining at 6, 12, and 24 h. Six C57 mice were used in each group. We then used the CX3CR1 + /GFP mice for MPO immunofluorescence with six mice per group. We used 40 μm-thick brain slices for immunohistochemistry. For iba1 staining, we used the ABC method. The antibodies were Rabbit anti iba1 (Solarbio Life Sciences, Beijing, China, 1:600), Goat anti Rabbit (Solarbio, 1:300), and streptavidin–horseradish peroxidase conjugate (STR-HRP). For MPO fluorescence staining, we used Rabbit anti MPO (Solarbio, 1:600) and Goat anti Rabbit (Solarbio, 1:300). Following each antibody staining, the brain slices were washed three times with PBST (PBS + Triton). Images of the immunohistochemically labeled sections and the fluorescently immunolabeled sections were obtained using an Olympus CKX41 fluorescence inverted microscope (Olympus, Tokyo, Japan). The images were captured in the FITC and TRITC channels using Cell-P imaging software (Olympus). When the two images were merged, the cells appeared yellow.
8
Detailed Microscopy and Spectroscopy Protocols
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The CKX41 fluorescence inverted microscope was purchased from Olympus (Tokyo, Japan), and the Mini-Protean Tetra and Mini Trans-Blot electrophoresis systems, Gel Doc XR imaging system and Model 680 microplate reader were purchased from Bio-Rad. The EPICS XL flow cytometer was obtained from Beckman Coulter (Miami, FL, USA) and the CO-150 CO2 incubator was purchased from New Brunswick Scientific (Edison, NJ, USA). The UV1000 UV-VIS spectrophotometer was purchased from Techcomp Limited (Shanghai, China).
9
Histological Evaluation of Rat Gastric Tissue
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The 4% formalin-fixed rat gastric tissues (25 ℃ for 24 h) underwent routine alcohol dehydration and paraffin embedding. The embedded tissues were cut into 5 µm slices and mounted on slides. After dewaxing and rehydrating, the slices were stained with H&E. The stained slices were dehydrated with graded ethanol and xylene. The glass slides were mounted with neutral balsam and covered with coverslips. The rat gastric tissues were imaged using the Olympus CKX-41 fluorescence inverted microscope (Olympus, Inc., Tokyo, Japan). The atrophy and inflammatory of gastric glands were scored by 2 pathologists according to the visual analog scale of the new Sydney system (18 (link),19 (link)).
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