For the invasion assay, 2×104 HeLa cells in FBS-free DMEM were placed in the upper chamber of an insert (8-µm pores; Merck KGaA). Matrigel (Merck KGaA) was employed to pre-coat the membrane of the Transwell chambers. The lower chambers were incubated in culture medium supplemented with 10% FBS for 24 h. Then, the HeLa cells on the upper surface were scraped and washed away. Subsequently, the cells on the lower surface were fixed and stained with Diff-Quik staining kit (Sysmex Corp.) at RT for 2 h. The invaded cells in the lower surface of membrane were also fixed and stained with Diff-Quik kit (Sysmex Corp.). The invaded cells were imaged and counted under a BX43 Upright Microscope (Olympus Corp.) at a magnification of ×200 from 10 random fields in each well. For the migration assay, 1.5×106 cells/well were seeded in 6-well plate for overnight culture until the cells reached an ~90% confluence. The scratch was generated using a 200-µl sterile pipet tip in the hood. After aspiration and washing, fresh complete medium was added. Then the cells were cultured for another 24 h. Cell migration was monitored under a BX43 Upright Microscope (Olympus Corporation) and images were captured at 0 and 24 h.
Bx43 upright microscope
Manufactured by Olympus
Sourced in Japan, United States
The BX43 is an upright microscope designed for a variety of laboratory applications. It features a sturdy, ergonomic design and provides high-quality optical performance. The microscope is equipped with a LED illumination system and can accommodate a range of objective lenses to enable detailed observation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Cellsens entry software, by Olympus (4 mentions)
Inform 2, by PerkinElmer (3 mentions)
Dp74 cmos camera, by Olympus (3 mentions)
Mantra snap software 1, by PerkinElmer (2 mentions)
Anti rabbit peroxidase conjugated secondary antibody, by Merck Group (2 mentions)
Aec substrate, by Abcam (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Cellsens standard software, by Olympus (2 mentions)
Szx7 stereomicroscope, by Olympus (1 mentions)
Dp72 camera, by Olympus (1 mentions)
Rnascope multiplex fluorescent v2 kit, by Advanced Cell Diagnostics (1 mentions)
Anti cd4, by BioXCell (1 mentions)
Anti cd8, by BioXCell (1 mentions)
Anti cd4, by Abcam (1 mentions)
Anti cd11c, by BioLegend (1 mentions)
Anti cd3, by Bio-Rad (1 mentions)
Anti foxp3, by Thermo Fisher Scientific (1 mentions)
Dp73 color camera, by Olympus (1 mentions)
Dako endogenous blocking reagent, by Agilent Technologies (1 mentions)
Neutral buffered formalin, by Avantor (1 mentions)
36 protocols using bx43 upright microscope
1
Cell Invasion and Migration Assays
2
Immunohistochemical Evaluation of UBQLN4 and MRE11A
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Endoscopic biopsy specimens and surgically resected ESCC tumors were stained with UBQLN4 monoclonal antibody (mAb; #sc‐136145; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and/or MRE11A polyclonal antibody (pAb; #4895S; Cell Signaling Technology, Danvers, MA, USA) as previously described [33 ]. Images were taken by the BX43 upright microscope (Olympus, Tokyo, Japan) with a magnification of 20× using the mantra snap Software 1.03 (Perkin Elmer, Waltham, MA, USA). H‐scores were calculated using the inform 2.4 software (Perkin Elmer) and following manual instructions. Briefly, the tumor area was segmented from the stromal area and nuclei/cytoplasm compartments were distinguished by detecting the intensity of hematoxylin and/or 3,3'‐diaminobenzidine (DAB) staining. The optical signal threshold to classify the score into 4‐bins was set to 0.05, 0.12, and 0.2. Five slides per case were analyzed and the average H‐score was then used as the final value. The cutoff values for UBQLN4 and MRE11A were determined by considering H‐score values for the mean value observed in normal adjacent epithelia esophagus tissues plus 10 SD.
3
Cardiac Tissue Analysis in Mice
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At the end of the 6th week of MET and 1 week of isoproterenol treatment, mice were anesthetized using isoflurane and euthanized by cervical dislocation. Hearts were immediately perfused with ice cold phosphate buffered saline, removed and appropriately stored for RNA, protein, biochemical, and histological analysis. Tissues stored in RNAlater were used for RNA isolation, and tissues were immediately flash frozen in liquid nitrogen for proteins. A small piece of the heart tissue (~20 mg) was immediately processed for GSH assay. Middle region of myocardial sections were embedded in paraffin and sectioned for histological evaluation. Slides were stained with hematoxylin and eosin to determine cardiac damage and picrosirius red (PSR) stain for collagen accumulation. Images were captured using an Olympus BX43 upright microscope.
4
Visualization of Transgenic Plant GUS Expression
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GUS staining analysis of transgenic young trees was performed as previously described (Jefferson et al., 1987 (link)). For each promoter::GUS, at least six independent transgenic lines were used for GUS staining analysis, and the experiments were repeated thrice. Images of the stained leaves were captured using a two-colour infrared laser imaging system (Odyssey, USA); and images of the roots, stems, and veins were captured using a BX43 upright microscope (Olympus, Japan); and images of apical buds were recorded using a SZX7 stereomicroscope (Olympus, Japan).
5
Quantifying Neural Cell Proliferation
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To quantify neurogenesis, Ki67 stained sections were imaged on an Olympus BX43 upright microscope with a DP-72 camera. As Ki67 is present in the nucleus of actively dividing cells, it was possible to count discrete puncta that represent dividing cells. These were counted by hand and normalized for ventricle length. This normalized cell number (with units of cells/μm) is used to quantify neurogenesis.
6
Histological Quantification of Intestinal Mast Cells and Eosinophils
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The small intestine was fixed with 4% paraformaldehyde and subsequently embedded in paraffin. Paraffin sections (4-μm-thick) were stained with toluidine blue for mast cell staining and hematoxylin and eosin (H&E) for eosinophil staining. Mast cell and eosinophil numbers in the small intestine were evaluated in 3 sections from at least ten mice. Representative microscopic images were obtained using a BX43 Upright Microscope (Olympus, Tokyo, Japan) at ×400 magnification based on a high-power field (HPF).
7
Melanoma Protein Expression Analysis
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All of the FFPE tissues analysed were provided by SJHC. The cohort consists of 80 FFPE tissues (primary melanomas (n = 22), metastatic stage III (n = 12) and metastatic stage IV (n = 46)) from melanoma patients. FFPE tissues from patients with nevus (n = 7) were collected as the normal control. The clinical information for the melanoma patients is described in Table S1 . IHC was performed as previously described,25 , 33 using the mouse anti‐human ILF2 Ab (1:250 dilution, Santa Cruz, Cat# sc‐365068) and U2AF2 Ab (1:200 dilution, Santa Cruz, Cat# sc‐53942). Images were taken by the BX43 upright microscope (Olympus, Tokyo) at 20× magnification and with the Mantra Snap Software 1.03 (Perkin Elmer, Waltham). The images were analysed using inForm 2.4 software (Perkin Elmer). H‐scores were calculated following the inForm software instructions available at https://www.perkinelmer.com/ Content/LST_Software_ Downloads/inFormUserManual_2_3_0_rev1.pdf.
8
RNA ISH Assay for Melanoma Progression
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All of the FFPE tissues analysed were obtained from the SJHC pathology department. The cohort consists of 55 FFPE tissues [primary melanomas (n = 18), metastatic stage III (n = 17) and metastatic stage IV (n = 20)] from melanoma patients. FFPE tissues from the nevus (n = 12) were also collected as the normal control. The clinical information for those patients is described in Table S1 . RNA ISH assays were processed according to the manufacturer's instructions and as previously reported.34 Tissue slides (5 μm) were stained with the Hs‐ILF2 RNA probe and/or U2AF2 RNA probe (Advanced Cell Diagnostics, Newark) using RNAscope Multiplex Fluorescent Kit V2 according to the manufacturer's instructions available at https://acdbio.com/technical‐support/user‐manuals . Images were taken using an Olympus BX43 upright microscope with 20× magnification and analysed by inForm 2.4 software to calculate the number of foci per cell.
9
SARS-CoV-2 Infection Inhibition Assay
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MDBK cells were plated on eight well glass slide and incubated overnight at 37 °C and 5% CO2. After incubation, cells were pre-treated for 1 h with CIGB-325 (30 μM) or vehicle (PBS) and infected with 100 µL of virus at a concentration of 70 000 TCID50/well. After 1h of incubation, final volume was completed up to 500 µL and the appropriate drug's concentration was maintained for 16 h and 24 h. Subsequently, the cells were washed with cold PBS three times and fixed in 4% formaldehyde for 10 min at 4 °C. After permeabilization with 0.5% Triton X-100 for 10 min, cells were blocked by incubation with 4% bovine serum albumin (Sigma, MO,USA) in PBS for 30 min at room temperature, washed again, and incubated with 30 μg/mL human polyclonal anti-SARS-CoV-2 (CIGB, Cuba) for 2 h at room temperature.
Then, peroxidase-conjugated anti-rabbit secondary antibody 1:100 (Sigma, MO, USA) was incubated for 1 h at room temperature and washed 3 times with PBS. Finally, AEC substrate (Abcam, Cambridge, United Kingdom) was added and coverglass was mounted using 40% glycerol mounting medium and analyzed using a BX43 upright microscope (Olympus, USA).
Then, peroxidase-conjugated anti-rabbit secondary antibody 1:100 (Sigma, MO, USA) was incubated for 1 h at room temperature and washed 3 times with PBS. Finally, AEC substrate (Abcam, Cambridge, United Kingdom) was added and coverglass was mounted using 40% glycerol mounting medium and analyzed using a BX43 upright microscope (Olympus, USA).
10
CIGB-325 Inhibits SARS-CoV-2 Infection in MDBK Cells
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MDBK cells were plated on eight-well glass slides and incubated overnight at 37 °C and 5% CO2. After incubation, cells were pre-treated for 1 h with CIGB-325 (30 μM) or vehicle (PBS) and infected with 100 µL of virus at a concentration of 70,000 TCID50/well (MOI = 0.1). After 1 h of incubation, final volume was completed up to 500 µL, and the appropriate drug’s concentration was maintained for 16 h and 24 h. Subsequently, the cells were washed with cold PBS three times and fixed in 4% formaldehyde for 10 min at 4 °C. After permeabilization with 0.5% Triton X-100 for 10 min, cells were blocked by incubation with 4% bovine serum albumin (Sigma, St Louis, MO, USA) in PBS for 30 min at room temperature, washed again, and incubated with 30 μg/mL human polyclonal anti- SARS-CoV-2 for 2 h at room temperature. Then, peroxidase-conjugated anti-rabbit secondary antibody 1:100 (Sigma, St. Louis, MO, USA) was incubated for 1 h at room temperature and washed 3 times with PBS. Finally, AEC substrate (Abcam, Cambridge, UK) was added and coverglass was mounted using 40% glycerol mounting medium and analyzed using a BX43 upright microscope (Olympus America Inc., Waltham, MA, USA).
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