Mice were deeply anesthetized with pentobarbital sodium (50 mg/kg, i.p.) and sequentially perfused 30 mL of 1 × phosphate-buffered saline (PBS) followed by 30 mL of 4% (w/v) paraformaldehyde (PFA). The brains were subsequently removed and post-fixed in 4% PFA at 4 °C overnight. After cryoprotection of the brains with 30% (w/v) sucrose, coronal sections (40 μm) were cut on a cryostat (Leica CM1860, Germany). Then the brain slices were rinsed three times with PBS and blocked with blocking buffer (10% goat serum in PBS with 0.2% Triton X-100) for 1.5 h at 37 ºC. Sections were incubated with anti-NeuN monoclonal antibody (Rabbit 1:2000, Abcam, ab177487, RRID: AB_2532109) at 4ºC for 48 h. After washing four times in PBS (each time for 10 min), sections were incubated with secondary antibody Alexa Fluor® 594 (Goat anti-Rabbit 1:500, Jackson ImmunoResearch Inc., 111–585-144, RRID: AB_2307325) for 1.5 h at 37ºC. Then the brain slices were rinsed with PBS three times before mounted on a glass slide with 70% glycerol in PBS. Fluorescent images were taken by a Nikon Eclipse Ni-E upright fluorescence microscope (4x, Nikon, Japan) and confocal microscope (20x, 40x, Nikon, Japan). As regards imaging analysis of quantification of neuron numbers, we used the Fiji software (https://imagej.net/Fiji ).
Eclipse ni e upright fluorescence microscope
Manufactured by Nikon
Sourced in Japan
The Eclipse Ni-E is an upright fluorescence microscope designed for advanced imaging applications. It features a modular design and supports a variety of fluorescence techniques, including widefield, confocal, and TIRF microscopy. The Ni-E provides high-resolution imaging capabilities and is suitable for a range of research and clinical applications.
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Lab products found in correlation
Ab177487, by Abcam (1 mentions)
Cm1860, by Leica (1 mentions)
Alexa fluor 594, by Jackson ImmunoResearch (1 mentions)
Deadend fluorometric tunel system, by Promega (1 mentions)
Gelatin, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Cell Signaling Technology (1 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
Anti sma α antibody, by BioLegend (1 mentions)
Hoechst, by Merck Group (1 mentions)
Alexa fluor 488 donkey anti mouse secondary antibody, by Jackson ImmunoResearch (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
Ym3038, by Abcam (1 mentions)
5 protocols using eclipse ni e upright fluorescence microscope
1
Immunohistochemical Labeling of Neurons
2
Myocardial Apoptosis Quantification
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Myocardial apoptosis was determined by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay kit (DeadEnd™ Fluorometric TUNEL system, Promega Corporation, Madison, WI, USA) according to the manufacturer's instructions. TUNEL-positive nuclei were stained green and DAPI nuclei were stained blue. Ten random fields were recorded for each heart. The apoptotic index was calculated as the percentage of the number of TUNEL-positive nuclei to the total number of DAPI nuclei. Images were obtained and analyzed using a Nikon Eclipse Ni-E upright fluorescence microscope (Nikon, Tokyo, Japan). Assessment was performed independently by two investigators blind to the experimental groups.
3
Transfection of mATFs with miR-125b
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Cover slips were coated with gelatin (Thermo Fisher Scientific) for 30 min at 37°C and washed once with sterile PBS. A total of 100,000 mATFs were seeded on the cover slip in each well of a 12-well plate. After 24 h, cells were transfected with miR-125b (125b) mimics or scrambled oligonucleotide (NC) with lipofectamine, as described earlier. After 2 days, cells were washed with PBS and fixed in 4% PFA for 10 min. Cells were blocked with 5% BSA with 0.2% triton for 60 min at room temperature and incubated with anti-SMA-α antibody (BioLegend) overnight at 4°C. Cells were washed 3 times with PBS containing 0.05% Triton before incubation with anti-mouse AF488 antibody (Biolegend, USA) for 60 min at room temperature. Cells were incubated with 5 µg/mL Hoechst 33,342 (Cell Signaling Technology, USA) in PBS for 10 min at room temperature and washed 3 times with PBS. The cover slips were mounted on glass slides with Prolong Diamond Antifade Mountant (Life Technologies). Images were taken at 20X using a Nikon Eclipse Ni-E upright fluorescence microscope. Images were analysed using ImageJ.
4
Detecting Cas9-HA Protein Expression
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MOLM13 cells were fixed with 4% paraformaldehyde (Sigma Aldrich) and adhered to glass slides using cytospin at 400 rpm for 3 min. The cells were blocked with 5% normal donkey serum (Jackson Immuno Research), permeabilized with 0.2% Triton X-100, and incubated overnight with a mouse anti-HA antibody (Clone F-7, Cat #7392, Cell Signaling Technology, 1:250 dilution) at 4 °C, washed three times with PBS and incubated with Alexa Fluor® 488 donkey anti-mouse secondary antibody (Jackson Immuno Research) for 1 h at room temperature. The cells were finally stained with Hoechst (Sigma Aldrich) for 5 min at room temperature and washed three times with PBS. In the EV uptake experiment (Fig. 1f ), the cells were stained with Hoechst only but not with any antibody. Images were captured using Nikon Eclipse Ni-E upright fluorescence microscope. Images were analyzed using ImageJ. The number of Cas9-HA positive nuclei were counted and normalized by the number of Hoechst positive nuclei in the same image. The average percentage of Cas9-HA positive cells was calculated from three samples in each treatment.
5
Immunofluorescence Staining of Primary MEFs
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Primary MEFs were seeded in chamber slides, washed with PBS and fixed with 3% PFA/PBS for 12 min at room temperature. Cells were permeabilized with 0.5% Triton solution (20 mM HEPES, 50 mM NaCl, 3 mM MgCl2, 300 mM Sucrose, 0.5% Triton X‐100) for 5 min, blocked with 5% normal goat serum for 1 hr and stained with primary antibodies overnight at 4°C. Cells were washed with PBS and stained with FITC/TRITC secondary antibodies. Samples were then incubated with DAPI for 1 min, washed with PBS and mounted with ProLong™ Gold Antifade Mountant (Invitrogen). Cells were imaged with Nikon Eclipse Ni‐E upright fluorescence microscope. Primary antibodies of HP1α (CST, 2616, 1:200), HP1β (CST, 8676, 1:800), H3 (Immunoway, YM3038, 1:10000), H3K9me3 (Abcam, Ab8898, 1:500), H3K27me3 (CST, 9733, 1:1000), H3K4me3 (Active Motif, 61379,1:500), H4K20me3 (Active Motif, 39,671, 1:500), Orc2 (gift from Bruce Stillman, 920‐4, 1:200) were used at indicated concentrations for immunofluorescence.
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