DLD-1/UHMK1-2 cells were seeded on coverslips for overnight with or without IL-6 and fixed with 4% paraformaldehyde for 10 min, followed by permeabilization with 0.1% Triton X-100 for 5 min. Cells were then rinsed three times with PBS and blocked by 10 % donkey serum for half an hour. After that, cells were incubated with FLAG tag antibody for UHMK1 (1:50) or STAT3 antibody (1:50) overnight. The Alexa Fluor 488 and AlexaFluor 594 secondary antibodies were then incubated for 1 h at room temperature. Coverslips were mounted on slides with DAPI. Cells were acquired and photographed on a confocal laser scan microscope (Zeiss, Germany).
Laser scan confocal microscope
Manufactured by Zeiss
Sourced in Germany
The Laser Scan Confocal Microscope is a high-resolution imaging system that uses a focused laser beam to scan a specimen and capture detailed images. It provides optical sectioning capabilities, allowing for the examination of specific focal planes within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 conjugated goat anti mouse igg, by Sangon (2 mentions)
Cy3 conjugated goat anti rabbit igg h l, by Wuhan Servicebio Technology (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Alexa fluor 488 goat anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Bx51 fluorescence microscope, by Olympus (1 mentions)
H e staining kit, by Sangon (1 mentions)
Cyto id autophagy detection kit, by Enzo Life Sciences (1 mentions)
Facsaria 2, by BD (1 mentions)
Facs arial 2 flow cytometer, by BD (1 mentions)
5 ethynyl 2 deoxyuridine (edu), by Thermo Fisher Scientific (1 mentions)
Er tracker red, by Beyotime (1 mentions)
Fluorescence conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Fluorescent microscope, by Olympus (1 mentions)
11 protocols using laser scan confocal microscope
1
Immunofluorescence Assay for UHMK1 and STAT3
2
Immunofluorescence Analysis of SMIM3 Expression
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The cells in T25 flasks were collected and washed several times with phosphate-buffered saline (PBS). Then the cells were fixed using 4% paraformaldehyde (PFA) for 20 min at room temperature (RT). Further, Triton-X-100 (Beyotime, Shanghai, China) was applied to permeabilized cells for 10 min, and nonspecific binding was blocked with 5% BSA (Solaibao Biotechnology, Beijing, China) for 30 min at RT. Followed by washing, cells were incubated overnight at 4 °C with diluted (1:100) primary anti-SMIM3 Polyclonal Antibody (Thermo Fisher Scientific, Waltham, MA, USA). The cells were then incubated with diluted (1:200) secondary antibody Cy3 conjugated Goat Anti-Rabbit IgG (H + L) (Servicebio, Wuhan, China) for 1 h at RT, followed by washing in PBS and staining with DAPI (Solaibao Biotechnology, Beijing, China). Analysis was conducted under a confocal laser scan microscope (Zeiss, Oberkochen, Germany).
3
Noninvasive PET Imaging of Tumor-Associated Macrophages
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PET scans were performed using a micro-PET/CT scanner (Inviscan, IRIS, Paris, France). [68Ga]Ga-DOTA-M2pep (3.7 MBq) was administered to the B16F10 tumor-bearing mice (n = 4) via the tail vein. At 30 min, 1 h, and 2 h post-injection, the tumor-bearing mice were imaged with the micro-PET/CT scan under anesthesia with 2% isoflurane. Each scan was completed within 15 min. For the blocking scans, B16F10 tumor-bearing mice (n = 3) were pretreated with free M2pep (10 mM) in 100 μL saline by intravenous injection 60 min before administration of [68Ga]Ga-DOTA-M2pep (3.7 MBq). Micro-PET/CT scans were acquired at 60 min post-injection as described above. The obtained images were reconstructed using three-dimensional ordered-subset expectation maximization (3D OSEM) and then processed using Osirix MD. Regions of interest (ROIs) were manually drawn over the tumor regions and organs, and quantitative analyses were calculated. Immunofluorescence staining was performed 24 h after micro-PET scan to determine the [68Ga]Ga-DOTA-M2pep targeting efficiency. The harvested tumors were fixed with paraformaldehyde for 24 h, then were frozen and sectioned at 10 μm, followed by treatment with Fitc-M2pep, and staining with M2 TAMs marker PE-CD206 antibody and DAPI. The localization of Fitc-M2pep and PE-CD206 signals was observed with a confocal laser scan microscope (Zeiss, Jena, Germany).
4
Cytostatic-Induced Apoptosis in Cancer Cells
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Efficacy of different cytostatic agents to induce death of cancer cells was initially assessed by immunocytochemistry. For this purpose, cells were plated on glass cover slips and treated with different concentrations of doxorubicin (0.1 μM to 10 μM), cisplatin (10 μM to 40 μM) or temozolomide (10 μM to 200 μM; all obtained from the University of Leipzig Medical Center Pharmacy) for 3 h to 24 h. Cells were subsequently fixed and stained with DAPI and antibodies against cleaved-caspase-3 as delineated below. Apoptotic cells were counted on a Zeiss confocal laser scan microscope and expressed as per cent of total number of cells present in the same observation fields. To assess effects of chemokines on apoptotic cell death, cells were treated with 100 ng/ml of CXCL11 and/or CXCL12 24 h prior to the addition of cytostatics. After another 6 - 12 h, apoptotic cells were identified by the BD FITC Annexin V Apoptosis Detection Kit (Becton Dickinson, Franklin Lakes, NJ) and 10.000 events were quantified on a Becton Dickinson LSRFortessa™ flow cytometer. Data were analyzed using the FlowJo version 10 software (FlowJo; Ashland, Oregon). Apoptotic cell numbers present in cytostatic-treated cultures were set to 1.
5
Histological and Immunohistochemical Analysis of Larval Tissues
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Larval tissues were isolated and fixed with 4% paraformaldehyde in PBS by incubating at 4 °C overnight. Fixed tissues were gradually dehydrated. Tissues were then embedded in melted paraffin and sliced into 7 μm sections using a paraffin slicing machine. Sections were adhered to gelatin-coated glass slides and slides were dried at 42 °C overnight, followed by dewaxing. Sections were gradually rehydrated and digested with 20 mM proteinase K at 37 °C for 10 min. For tissue HE staining, the sections were then stained using an HE staining kit (Sangon, Shanghai, China) according to the manufacturer’s instructions. Positive staining signals were observed with an Olympus BX51 fluorescence microscope (Olympus, Shinjuku-ku, Japan). For immunohistochemical assay, the sections were blocked with 5% BSA at 37 °C for 30 min and then incubated with anti-caspase-3 antiserum (1: 200) at 4 °C overnight. The slides were washed six times and incubated with Alexa Fluor-488 goat anti-rabbit secondary antibody (Life Technologies) at 37 °C for 1 h. DAPI (1: 1000) was used for nuclei staining, followed by observation with a Laser Scan Confocal Microscope (Carl Zeiss LSM 700 model, Zeiss).
6
Immunofluorescence Localization of rCgPERK in Oyster Hemocytes
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Hemocytes from oyster were collected and washed two times using modified L15 medium (M-L15, supplemented with KCl 0.54 g L−1; CaCl2 0.6 g L−1; MgSO4 1.0 g L−1; MgCl2 3.9 g L−1; NaCl 20.2 g L−1) [18] . The cells were fixed by 4% paraformaldhyde for 20 min. After blocking with 20 µL of 3% BSA for 1 h, the sample was incubated with anti-rCgPERK antibody (1:1000) as first antibody and Alexa Fluor 488-conjugated anti-mouse IgG (1:2000) as secondary antibody at 37 °C for 1 h. The serum from pre-immunization mice was used as negative control. The nucleus was stained blue by 4′,6-Diamidino-2-Phenylindole (DAPI). After the last three times washing, the hemocytes were observed and captured by using Laser Scan Confocal Microscope (Carl Zeiss, Germany).
7
Quantifying Autophagy in Hemocytes
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The percentages of hemocytes with autophagy activity were quantified using the commercial CytoID® autophagy detection kit (ENZO Life Science, ENZ-51, 031-0050) following the manufacture's instruction. Hemolymph was collected with modified Alsever's solution (MAS, glucose 20.8 g L−1; EDTA 3.36 g L−1; NaCl 22.5 g L−1; sodium citrate 8.0 g L−1; pH 7.4) at the ratio of 1:1, and then centrifuged immediately at 600 g, 4 °C for 10 min. After washing with modified L15 medium twice, the pellet containing hemocytes was suspended in 100 µL CytoID® green detection reagent (diluted with 1:2000 in modified L15 medium, v/v), and incubated in the dark at room temperature for 90 min. After thoroughly washing with modified L15 medium for two times, hemocytes were incubated with Hoechest 33342 Nuclear Stain (1mM diluted 1:1000 in modified L15 medium) at room temperature for 20 min. After the last two times of washing, the hemocytes were re-suspended in 100 µL modified L15 medium, and observed and captured by using Laser Scan Confocal Microscope (Carl Zeiss, Germany). The autophagosome rate of hemocytes in each group was analyzed by FACSAria II flow cytometry (BD Biosciences, USA). The percentage of hemocytes with autophagosomes was calculated, and the ratio of percentage between tested group and control group was used to determine the autophagy activity.
8
EdU Labeling of Oyster Gill Cells
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EdU labeling assay was performed as previously described (43 (link)). In brief, six oysters were cultured with sterile seawater with EdU (Life technologies, 2 mg L-1) in an aerated tank for 48 h. Cross sections of gill were made as described above. The slides were then fixed with 4% PFA at room temperature for 15 min. After three times washing with 3% BSA in TBS, 0.1% Triton® X-100 in TBS was used to treat the samples at room temperature for 10 min. CgDM9CP-4 positive cells were stained as mentioned above and after the final three times washing with TBS (containing 3% BSA), 0.1% Triton® X-100 in TBS was used to treat the samples at room temperature for 10 min. Samples were then incubated with the 1 × Click-iT™ Reaction Buffer (provided with the kit) for 30 min, and washed thoroughly with PBS (3 × 15 min). EdU were analyzed on an FACS Arial II flow cytometer (Becton, Dickinson and Company, USA). For the new generated hemocytes in gill, slices were incubated with DAPI (diluted 1:1,000 in PBST) for 5 min, washed extensively with PBS, mounted with 80% glycerin, and monitored under a Laser Scan Confocal Microscope (ZEISS).
9
Subcellular Localization of CgTRPV4 in Oyster Haemocytes
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Immunofluorescence assay was carried out to investigate the subcellular localization of CgTRPV4 in haemocytes as previously described with some modification [34] (link). The haemocytes were collected from five oysters, re-suspended in L15 cell culture media, deposited on dishes precoated with poly-Lysine (a drop on each) in the wet chamber, and fixed with 4% paraformaldehyde. After blocked with 3% bovine serum albumin (BSA) at room temperature for 30 min, the samples were incubated with anti-rCgTRPV4 (diluted 1:1000 (v/v) in 3% BSA) antibody at 37°C for 1 h. Alexa Fluor 488-conjugated goat anti-mouse IgG (Sangon, diluted 1:1500 in 3% BSA) was used separately to incubate with the samples at 37°C for 1 h. ER-Tracker Red (Beyotime, diluted 1:1500 in 3% BSA) and Dil (Beyotime, diluted 1:1000 in 3% BSA) were used to incubate with the samples to stain ER or the membrane. After incubated with 4, 6-diamidino-2-phenylindole hydrochloride (DAPI, diluted at 1:500 with 3% BSA) for 10 min to stain the nuclei, fluorescent images of the oyster haemocytes were obtained using a Laser Scan Confocal Microscope (ZEISS).
10
Subcellular Localization of PyGRP78 in Scallop Haemocytes
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Immunofluorescence assay was carried out to investigate the subcellular localization of PyCNX in haemocytes as previously described with some modification [30] (link). The haemocytes were collected from five scallops, re-suspended in L15 cell culture media, deposited on dishes precoated with poly-Lysine (a drop on each) in the wet chamber, and fixed with 4% paraformaldehyde. After blocked with 3% bovine serum albumin (BSA) at room temperature for 30 min, the samples were incubated with anti-PyCNX (diluted 1:1000 (v/v) in 3% BSA) and commercial anti-GRP78 (ER marker molecule) (Beyotime, diluted 1:1000 in 3% BSA) antibodies at 37 °C for 1 h. The commercial anti-GRP78 antibody is produced by immunizing rabbits with a synthetic peptide corresponding to human GRP78 which shares high identity of 81% with that of PyGRP78. Alexa Fluor 488-conjugated goat anti-mouse IgG (Sangon, diluted 1:1500 in 3% BSA) and Alexa Fluor 555-conjugated goat anti-rabbit IgG (Sangon, diluted 1:1500 in 3% BSA) were used separately to incubate with the samples at 37 °C for 1 h. After incubated with 4, 6-diamidino-2-phenylindole hydrochloride (DAPI, diluted at 1:500 with 3% BSA) for 10 min to stain the nuclei, the fluorescent images of the scallop haemocytes were obtained using a Laser Scan Confocal Microscope (ZEISS).
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