Expression levels of NLRP1 and NeuN were detected by IF staining. A paraffin-embedded tissue section (5 mm thickness) was dewaxed using xylene and dehydrated using graded concentrations of alcohol and incubated with 3% H2O2 for inhibiting endogenous peroxidase activity (24 (link)). After blocking in 10% goat serum for 10 min at room temperature, the section was incubated with the primary antibodies in a blocking solution overnight at 4°C (24 (link)). Then, the slides were washed in PBS, followed by treatment with HRP-labeled anti-rabbit IgG (1:100; Beyotime, China) for 30 min at 37°C and washing with PBS. Images were acquired with a Nikon Eclipse Ni inverted microscope (TE2000, Nikon, Tokyo, Japan).
Eclipse ni inverted microscope
Manufactured by Nikon
Sourced in Japan
The Nikon Eclipse Ni is an inverted microscope designed for versatile imaging applications. It features a stable and vibration-resistant optical system, and can accommodate a range of accessories and objectives to support various sample types and magnification requirements.
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Lab products found in correlation
Hrp labeled anti rabbit igg, by Beyotime (2 mentions)
Wl02508, by Wanlei (2 mentions)
Vdac1, by Santa Cruz Biotechnology (1 mentions)
Dp73 type microscope, by Olympus (1 mentions)
3 3 diaminobenzidine (dab), by Solarbio (1 mentions)
Ab3683, by Abcam (1 mentions)
As014, by ABclonal (1 mentions)
Horseradish peroxidase hrp labeled anti rabbit igg, by Beyotime (1 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Corning (1 mentions)
Moflo xdp cell sorter, by Beckman Coulter (1 mentions)
9 protocols using eclipse ni inverted microscope
1
Immunofluorescence Staining of NLRP1 and NeuN
2
Cell Viability Assessment using PI-Hoechst Assay
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Cell viability was assessed using the PI-Hoechst assay (Beijing Solarbio Science & Technology Co., Ltd), according to the manufacturer’s instruction. Cells were observed using fluorescence microscopy at x400 magnification. Images were acquired with a Nikon Eclipse Ni inverted microscope (TE2000; Nikon Corporation).
3
Evaluation of Kidney Autophagy Markers
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Expression of Beclin-1, LC3, p62 and Cyt C in kidney tissues was detected by IHC staining. Paraffin-embedded kidney tissue fraction (5 μm thickness) and xylene dewaxed, then dehydrated using graded concentrations of alcohol, and incubation with 3% H2O2 inhibited endogenous peroxidase. After blocked in 10% goat serum for 10 min at room temperature, incubated with the primary antibody in blocking solution overnight at 4°C. The primary antibodies for IHC were p62 (1:50, sc-48402), Beclin-1 (1:200, WL02508, Wanlei Bio), MAPLC3B (1:50, A11280), and Cyt C (1:200, WL02410). After slides were washed in PBS and applied HRP-labeled anti-rabbit IgG (1:100; Beyotime, China) for 30 min at 37°C, then washed with PBS again. Staining was visualized by reaction with 3,3'-diaminobenzidine (DAB) (Solarbio, Beijing, China) and counterstaining with hematoxylin. Sections were made transparent with xylene. Finally, using the DP73 type microscope (Olympus, Japan) observed sections. We also performed IF co-staining using PINK1 (1:50, sc-517353) and VDAC1 (1:50, sc-390996, Santa Cruz Biotechnology, USA) antibodies to delineate the mitophagy in the kidney. Then images were acquired with a Nikon Eclipse Ni inverted microscope (TE2000, Nikon, Tokyo, Japan).
4
Immunohistochemical Detection of MPO
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The expression of MPO was detected by IF staining. Paraffin‐embedded tissue sections (5 mm thickness) were dewaxed in xylene, then dehydrated using graded concentrations of alcohol, and incubated with 3% H2O2 to inhibit endogenous peroxidase. After being blocked in 10% goat serum for 10 minutes at room temperature, they were incubated with the primary antibody (anti‐MPO 1:200, ab3683, Abcam) in a blocking solution overnight at 4°C. After slides were washed in PBS, HRP‐labeled anti‐rabbit IgG (1:100; AS014, ABclonal) was applied for 30 minutes at 37°C, and then washed with PBS again. Then, images were obtained with a Nikon EclipseNi inverted microscope (TE2000, Nikon).
5
Immunofluorescence Assay for NLRP3 and TOM20
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The 3μm thick paraffin sections were deparaffined, rehydrated, and prepared for immunofluorescence assays according to a standard protocol. The sections were incubated with primary antibodies as follows: anti-NLRP3 (WL02635, Wanlei, Shenyang, China) diluted 1:200 and anti-TOM20 (A19403, ABclonal, Wuhan, China) diluted 1:100 overnight at 4°C. After being washed with PBS 3 times, the sections were incubated with secondary antibody. The sections were washed with PBS 3 times again and then sealed with coverslips. Fluorescence images were acquired with a Nikon Eclipse Ni inverted microscope (TE2000; Nikon, Tokyo, Japan).
6
Beclin-1 Expression by Immunofluorescence
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Expression of Beclin-1 was detected by IF staining. Paraffin-embedded tissue fraction (5 mm thickness) and xylene dewaxed, then dehydrated using graded concentrations of alcohol, and incubated with 3% H2O2 inhibited endogenous peroxidase.17 (link) Afterwards, it was blocked in 10% goat serum for 10 min at room temperature, incubated with the primary antibody in blocking solution overnight at 4°C. The primary antibodies for Beclin-1 (1:200, WL02508, WanleiBio) were used. Afterwards, the slides were washed in phosphate buffer saline (PBS), applied with horseradish peroxidase (HRP)-labeled anti-rabbit IgG (1:100; Beyotime, China) for 30 min at 37°C, then washed with PBS again. Then, images were acquired with a Nikon EclipseNi inverted microscope (TE2000, Nikon, Tokyo, Japan).
7
Immunofluorescence Staining of Txk Expression
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The expression level of Txk was detected by IF staining as previously described (13 (link)). In brief, paraffin-embedded tissue fraction (5-mm thickness) was dewaxed with xylene, then dehydrated using graded concentrations of alcohol, and incubated with 3% H2O2 inhibitor, endogenous peroxidase. After blocking in 10% goat serum for 10 min at room temperature, the tissues were incubated with the primary antibody of Txk (1:100) in blocking solution overnight at 4°C. Then, the slides were washed in PBS and HRP-labeled anti-rabbit IgG was applied for 30 min at 37°C, followed by a PBS wash. Finally, images were acquired with a Nikon EclipseNi inverted microscope (400× magnification; TE2000; Nikon Corporation).
8
Immunohistochemical Analysis of Muscle Fiber Types
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rNLS8 and nTg mice were perfused with ice-cold PBS followed by 10% (v/v) neutral buffered formalin. The hindlimb muscles were collected, washed in PBS overnight and processed in a 10–30% sucrose gradient for cryoprotective embedding. The muscles were cross-sectioned at 10 µm thickness, incubated in 5% FBS in PBS for blocking, and then immunostained using the following primary antibodies: mouse anti-BA-D5 (for myosin heavy chain type I; 2–5 µg/mL, DSHB, U. Iowa), mouse anti-BF-F3 (for myosin heavy chain type IIB; 2–5 µg/mL, DSHB, U. Iowa), mouse anti-SC-71 (for myosin heavy chain type IIA; 2–5 µg/mL, DSHB, U. Iowa) [36 (link)]. After overnight incubation, tissue sections were washed and then incubated with Alexa Fluor secondary antibodies (1:1000, Molecular Probes) for 2 h. Sections were imaged using a Nikon Eclipse Ni inverted microscope.
9
Isolation and Characterization of BMPR1a Macrophages
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The BMPR1a cell lines were derived from 5 month old male C57Bl6 mice with the following genotypes:
LysMCre+.BMPR1awt/wt.mTmG+/− (Control) will be referred to as PODS4 and LysMCre+.BMPR1aflox/flox .mTmG+/− (cKO) will be referred to as PODS5. Femur and tibia bones were harvested from mice, sliced lengthwise and placed in a T-75 vented tissue culture treated flask (Greiner) with 20 mL of DMEM (Corning) supplemented with 10% FBS (Seradigm) and 3X antibiotic (Gibco). Cells were cultured in a humidified incubator at 37°C and 5% CO2. Media was changed at 48 h, then the bone fragments were removed between day 7 and 10 of culture. Cells were expanded into two flasks prior to flow cytometry sorting. Sorting was performed by the CU Cancer Center Flow Cytometry Shared Resource using a MoFlo XDP Cell Sorter (Beckman Coulter) with a 100 μm nozzle tip. After multiple flow cytometry sorts, PODS4 double positive cells expressing tdTomato and EGFP were collected, and PODS5 single positive cells expressing EGFP were collected. Both cell lines were expanded for macrophage polarization experiments. Cell morphology and EGFP expression was assessed at 20X on an Eclipse Ni inverted microscope (Nikon) (Supplemental Figure 3B ).
LysMCre+.BMPR1awt/wt.mTmG+/− (Control) will be referred to as PODS4 and LysMCre+.BMPR1aflox/flox .mTmG+/− (cKO) will be referred to as PODS5. Femur and tibia bones were harvested from mice, sliced lengthwise and placed in a T-75 vented tissue culture treated flask (Greiner) with 20 mL of DMEM (Corning) supplemented with 10% FBS (Seradigm) and 3X antibiotic (Gibco). Cells were cultured in a humidified incubator at 37°C and 5% CO2. Media was changed at 48 h, then the bone fragments were removed between day 7 and 10 of culture. Cells were expanded into two flasks prior to flow cytometry sorting. Sorting was performed by the CU Cancer Center Flow Cytometry Shared Resource using a MoFlo XDP Cell Sorter (Beckman Coulter) with a 100 μm nozzle tip. After multiple flow cytometry sorts, PODS4 double positive cells expressing tdTomato and EGFP were collected, and PODS5 single positive cells expressing EGFP were collected. Both cell lines were expanded for macrophage polarization experiments. Cell morphology and EGFP expression was assessed at 20X on an Eclipse Ni inverted microscope (Nikon) (
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