Neutrophils (5×105) were seeded into IBIDI™ µ-slide 8-well chamber slides, pre-incubated for 1 h with ruboxistaurin (200 nM), and NETosis was induced by LPS and PMA as described above. Cells were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature. Wells were blocked and permeabilised with buffer containing 5% bovine serum albumin (BSA), 0.1% saponin and 5% normal goat serum, and then stained with a rabbit anti-MPO (A0398) primary antibody for 90 min at 37°C. A secondary goat anti-rabbit Alexa Fluor® 594 antibody (ab150088) was added for 45 min at 37°C. ProLong™ Gold Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher) was used to stain DNA. Samples were imaged using a NIKON Widefield fluorescence microscope, using the 40× oil immersion objective lens. The DAPI (excitation/emission 395/455 nM) and Texas Red (excitation/emission 555/605 nM) filter sets were used for fluorescent imaging. Images were constructed using FIJI image analysis software, and the background was subtracted for the DAPI channel using FiJI.
Widefield fluorescence microscope
Manufactured by Nikon
The Widefield fluorescence microscope is a laboratory instrument designed to visualize and analyze fluorescently labeled samples. It utilizes a wide field of view to capture images of multiple cells or specimens simultaneously. The core function of this microscope is to provide a comprehensive overview of fluorescent signals within the sample.
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Lab products found in correlation
Evos fl auto cell imaging system, by Thermo Fisher Scientific (2 mentions)
Prolong gold antifade mountant with 4 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
Nucblue, by Thermo Fisher Scientific (1 mentions)
Hne11 s, by Alpha Diagnostic (1 mentions)
Magnetic plate, by Ozyme (1 mentions)
Neuromag paramagnetic nanobeads, by Ozyme (1 mentions)
Picrosirius red, by Polysciences (1 mentions)
Trichrome, by Merck Group (1 mentions)
Prolong, by Thermo Fisher Scientific (1 mentions)
7 protocols using widefield fluorescence microscope
1
Visualizing Neutrophil Extracellular Traps
2
Investigating Cyanobacteria-nTiO2 Hetero-Aggregation
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To investigate the hetero-aggregation between nTiO2 and Prochlorococcus, a 200 μL sample was collected from the bottom of culture flasks from one replicate of each treated group during medium-term experiments (section 2.5). This sub-sample was stained with 1X SYBR Gold nuclear stain (ThermoFisher) and imaging was carried out at 40× magnification using a Nikon widefield fluorescence microscope under brightfield and GFP fluorescence. Images were captured from both channels and subsequently merged to assess the presence and extent of aggregation between cyanobacterial cells and nTiO2. Controls containing nTiO2 only (100 mg L−1) were included to prove the nanoparticles did not get stained by the dye. In addition, NSW samples and untreated Prochlorococcus culture in the absence of nTiO2 were imaged to confirm aggregates were indeed nTiO2 rather than other particulate material.
3
Quantifying Oxidative Stress in CCI-Induced Brain Injury
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Brain samples were collected 24 h post-CCI from the mtD2g mice and were formalin-fixed and paraffin-embedded. Coronal brain sections were antigen-retrieved in citrate buffer, permeabilized, and blocked together in 0.2% Trion X-100 in TBST + 1%BSA and 10% normal horse serum for one hour at room temperature (RT). Then, the sections were incubated overnight at 4 °C with primary antibody 4-HNE antibody (1:250; HNE11-S, alpha diagnostic international, San Antonio, TX, USA) in 50% diluted blocking buffer. Alexa flour 594 donkey anti-rabbit (1:500; A212207, Invitrogen) was used as a secondary antibody at RT for 1 h. After washing, the samples were mounted on glass slides using a Prolong Glass antifade mount with Nucblue (P36981, Invitrogen, Waltham, MA, USA). Images were acquired using a Nikon widefield fluorescence microscope with NIS-Elements version 5.30.05. The 4-HNE mean intensity was measured using ImageJ software (n = 3/group).
4
Tracking Mitochondrial Dynamics in Primary Motor Neurons
5
Histological Characterization of Explanted Grafts
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Explanted grafts were collected and fixed in 4% PFA for 24 h and dehydrated in 30% sucrose for 48 h. Tissue samples were embedded in optimal cutting temperature (OCT) and 8 μm sections were placed on TOMO adhesive slides for immunostaining. Slides were stained with H&E (StatLab), picrosirius red (Polysciences), and tri‐chrome (Sigma) staining kits. Images for H&E and trichrome staining were obtained on a brightfield microscope, and picrosirius red staining was visualized using a circular polarized light microscope. For immunostaining, slides were fixed in 95% methanol and permeabilized in 0.1% Triton X‐100. Sections were treated with primary antibodies against CD31 (R&D systems AF3628) diluted 1:50, vWF (Millipore AB7356) diluted 1:10, and C‐peptide (DSHB GN‐ID4) diluted 1:200, overnight at 4 C. Samples were rinsed with PBS followed by 1 h room temperature incubation with 1:500 dilution of secondary antibody (AF 546, Sigma) and/or Alexa Fluor 647‐conjugated glucagon antibody (Novus Biologics IC1249R) diluted 1:2000. Samples were then rinsed with PBS, incubated with Hoechst 3322 for 5 min, and mounted using anti‐fade mounting medium (ProLong, LifeTechnologies). Images were obtained using a NIKON widefield fluorescence microscope.
6
Ligament Tissue SDS Treatment Imaging
7
Ligament Tissue SDS Treatment Imaging
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Ligament tissue sections treated with predetermined concentrations of SDS were scanned using an automated Nikon wide-field fluorescence microscope (4 × objective lens, 488 nm argon gas laser). The sections of UBM stained with CF-CHP and anti-collagen I antibody were imaged by an EVOS FL auto cell imaging system (Thermo Fisher Scientific) with a 20 × objective lens (GFP light cube for CF-CHP and RFP light cube for anti-collagen I antibody). Images within each experiment were obtained under identical microscope settings (e.g., exposure time, gain) to allow direct comparison.
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