Sections of formalin‐fixed, paraffin‐embedded tissues, xenografts, and ALI organotypic cultures were stained by standard HE staining. For immunohistochemistry staining, slides were subjected to antigen retrieval in citrate buffer (pH 6.0, Sigma‐Aldrich) at 95 °C for 20 min, and a blocking procedure was performed overnight with 5% bovine serum albumin (Sigma‐Aldrich,) and 0.05% Triton X‐100 (Sigma‐Aldrich) in DPBS(‐) (Gibco) at 4 °C. Primary antibodies used in this study included antibodies against MUC2 (Santa Cruz Biotechnology, SC‐15334, 1:500), KI67 (Thermo Fisher Scientific, 550609, 1:1000). Then, sections were stained using DAB Substrate Kit (#550880, BD). Images were acquired by Olympus IX73 microscopy.
Ix73 microscopy
Manufactured by Olympus
Sourced in Japan
The IX73 is an inverted microscope system designed for a variety of advanced imaging applications. It features a modular design and supports a range of objective lenses and complementary equipment to enable versatile microscopy techniques.
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Lab products found in correlation
Triton x 100, by Merck Group (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (2 mentions)
Cm1950 microtome, by Leica camera (2 mentions)
Fv1000 confocal laser scanning microscope, by Olympus (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Citrate buffer, by Merck Group (1 mentions)
Dab substrate kit, by BD (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Mouse anti ki67, by Cell Signaling Technology (1 mentions)
Formaldehyde fa, by Merck Group (1 mentions)
Anti casr, by Thermo Fisher Scientific (1 mentions)
E cadherin, by R&D Systems (1 mentions)
Chromogranin a, by Abcam (1 mentions)
Villin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Ab32376, by Abcam (1 mentions)
Ab68428, by Abcam (1 mentions)
Ab5417, by Abcam (1 mentions)
15 protocols using ix73 microscopy
1
Immunohistochemical Analysis of Tissue Samples
2
Visualizing Cd-Induced Intracellular Ca2+ Flux
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To visualize the effect of Cd on intracellular Ca2+ levels in renal cells, mRTEC cells were seeded at a density of 2 × 104 cells/well in 24-well plates. Next day, the cells were loaded with 1 μM Fluo-3/AM in the RMPI 1640 medium (phenol red free) for 30 min at 37 °C in the dark. After dye loading, the cells were washed twice with the medium. Then, the cells were pre-treated with inhibitors for 30 min, i.e. U73122 (1 μM), 2-APB (50 μM), and BAPTA/AM (10 μM), then added CdCl2 (5 μM) or CdCl2 (5 μM) + R467 (1 μM) for 1 hour. Finally, calcium imaging was observed by Olympus IX73 microscopy (Japan).
3
Ki-67 Expression in mRTEC Cells
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To detect the expression of Ki-67, the mRTEC cells were treated with CdCl2 (5 μM) with or without R-467 (1 μM), or S-467 (1 μM) for 24 h, or pre-treated with PD98059 (10 μM), SB202190 (10 μM) for 30 min. The cells were fixed for 30 min in 4% Formaldehyde (FA, Sigma-Aldrich). Then, the cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 20 min. After blocked with 3% normal goat serum, the cells were incubated with mouse anti-Ki-67 (1:100, Cell Signaling Technology) antibody overnight at 4 °C, followed by 1 h incubation with Alexa Fluor 488 goat anti-mouse IgG (1:200, Molecular Probes, Invitrogen). After washing twice with PBS, cell nucleus was stained by the DAPI (Invitrogen) for several minutes. The cells were washed in PBS for 10 min and the cells were mounted, then examined by Olympus IX73 microscopy (Japan). To detect the expression of CaSR (anti-CaSR, 1:40, Thermo fisher Scientific) on the membranes of HK-2 and mRTEC cells, the process was performed without the permeabilization step.
4
Immunohistochemical Analysis of PCNA
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Paraffin-embedded tumor sections (4 μm) were deparaffinized, rehydrated and then treated with of Proteinase K (20 mg/ml), followed by the addition of hydrogen peroxide (3%) to block the endogenous peroxidase activity. Thereafter, the section was incubated with anti-PCNA antibody at a 1:400 dilution for 1 h and detected with GTVisin™ anti-mouse/anti-rabbit immunohistochemical analysis kit. Finally the images were visualized under a IX73 microscopy (Olympus, Tokyo, Japan).
5
Cotyledon Leaf Vein Imaging Protocol
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Leaf vein patterning was imaging according to a previously described procedure with minor modifications106 . Briefly, cotyledons were fixed in 100% ethanol:acetic acid (6:1, v/v) overnight at 4 °C. They were then washed once in 100% ethanol and again in 70% (v/v) ethanol, followed by clearing in chloral hydrate solution (chloral hydrate/glycerol/water (8:1:3)) for 1 h at RT. Then, cotyledons were rinsed twice in water. Cotyledons were cleared again in 85% (w/v) lactic acid for at least 3 days at RT. Cotyledons were placed in lactic acid and observed using IX73 microscopy (OLYMPUS).
6
Immunohistochemical Staining Protocols
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For DAB staining of paraffin sections, 5 µm slices were fixed in 10% formaldehyde followed a protocol as described 61 (link). For Immunofluorescence staining of cryostat sections, 30 µm frozen slices were sectioned by a Leica CM1950 microtome and followed a protocol as previously described 61 (link). All antibodies used in this study were listed (Table S1 ). Images were analyzed by Olympus IX73 microscopy.
Images of cellular autophagic flux analysis were captured by the Olympus FV1000 laser scanning confocal microscope. For quantitative analysis of the fluorescence intensity, the integral optical density was measured by using Image-Pro Plus 6.0 software.
Images of cellular autophagic flux analysis were captured by the Olympus FV1000 laser scanning confocal microscope. For quantitative analysis of the fluorescence intensity, the integral optical density was measured by using Image-Pro Plus 6.0 software.
7
Immunohistochemical Analysis of Tissue Samples
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Sections of formalin-fixed, paraffin-embedded tissues, xenografts and ALI tissues were stained by standard HE staining. For immunohistochemistry and immunofluorescence staining, slides were subjected to antigen retrieval in citrate buffer (pH 6.0, Sigma-Aldrich) at 95 °C for 20 min, and a blocking procedure was performed overnight with 5% bovine serum albumin (BSA, Sigma-Aldrich,) and 0.05% Triton X-100 (Sigma-Aldrich) in DPBS(-) (Gibco) at 4 °C. Primary antibodies used in this study included antibodies against MUC2 (Santa cruz biotechnology, 1:200) and E-Cadherin (R&D Systems, 1:200), Vimentin (Cell Signaling Technology, 1:200), CD45(Invitrogen, 1:500), Chromogranin A (Abcam, 1:200), Villin (Cell Signaling Technology, 1:200). Secondary antibodies were either Alexa Fluor-488 or Alexa Fluor-594 Donkey anti-goat/mouse/rabbit IgG (Thermo Fisher Scientific). Images were acquired by Olympus IX73 microscopy.
8
Immunohistochemical Analysis of Retinal Tissues
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Retinal tissues were cryoprotected in 30% sucrose for 24 h and embedded in OCT medium (Thermo Scientific, Cat# 6502). Ten-micrometer tissue sections were cut at -20°C in a cryostat (Thermo Scientific) and collected on the poly-L-lysine coated slides. After blocking with 5% BSA and 0.1% Triton X-100 in PBS, the retinal sections were incubated overnight at 4°C with the following primary antibodies: GFAP (1:200, Abcam Cat# ab68428, RRID: AB_1209224), NeuN (1:300, Abcam Cat# ab177487, RRID: AB_2532109), Calretinin (1:500, Santa Cruz Biotechnology Cat# sc-365956, RRID: AB_10846469), Calbindin (1:200, Santa Cruz Biotechnology Cat# sc-365360, RRID: AB_10841576), Rhodopsin (1:400, Abcam Cat# ab5417, RRID: AB_304874) and PKCα (1:400, Abcam Cat# ab32376, RRID: AB_777294). The retinal sections were washed and incubated for 3 h at room temperature with the fluorophore-conjugated secondary antibodies. The retinal sections were observed using an Olympus IX-73 microscopy and the fluorescent signals were analyzed by Image J.
9
Immunostaining of T. gondii-infected cells
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BMDMs or HFFs were challenged by T. gondii tachyzoites (MOI = 2) for 12 h and were fixed with 4% paraformaldehyde. Cells were permeabilized with 0.5% TritonX-100 in PBS. After blocking with 2% BSA, cells were incubated with anti-SAG1 (cat no., AM316809U-N) for 16–24 h, and stained with Coralite594-conjugated goat anti-mouse IgG(H+L) (cat no., SA00013-3) for 1 h and DAPI. Images were acquired on Olympus IX73 microscopy.
10
EdU Cell Proliferation Assay
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For the EdU cell proliferation assay, approximately 1 × 105 cells per well were seeded and cultured in 12‐well plates. After indicated treatments, NRK‐49Fs were incubated with EdU solution (Cat#C10338, RiboBio, Guangzhou, China) for 2 h and then stained with Hoechst 33342. Images were collected by an Olympus IX73 microscopy (Olympus). The EdU incorporation rate was quantified by normalizing the number of EdU positive cells against total nucleus number. At least 800 cells were viewed and counted in each well.
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