Paraffin-embedded mouse lung sections were incubated with primary antibodies against PFKFB3 (A5593, Bimake) at 4 C overnight. Then, the sections were incubated with an HRP-conjugated secondary antibody (GK500710, Gene Tech) for 30 min followed by DAB solution for seconds. Next, the nuclei were stained with hematoxylin and the sections were sealed with neutral gum. Images were captured by microscopy.
A5593
Manufactured by Selleck Chemicals
Sourced in United States
A5593 is a laboratory equipment product. It is a device used for the purpose of performing scientific analysis and measurements in a controlled laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
F6057, by Merck Group (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Gk500710, by Gene Tech (1 mentions)
A0562, by Beyotime (1 mentions)
A0521, by Beyotime (1 mentions)
Mab5580 sp, by R&D Systems (1 mentions)
Fdm007, by Fdbio (1 mentions)
A5224, by Selleck Chemicals (1 mentions)
Am1021b, by Abcepta (1 mentions)
Ab260043, by Abcam (1 mentions)
A5202, by Selleck Chemicals (1 mentions)
Imager d2, by Zeiss (1 mentions)
Gk500705, by Agilent Technologies (1 mentions)
5 protocols using a5593
1
Immunohistochemical Analysis of PFKFB3 in Mouse Lungs
2
Immunohistochemistry of Lung Tissue
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Tissue samples were sectioned, deparaffinized, and processed for staining. The tissue was incubated with primary antibodies overnight at 4°C. Then lung sections were stained with FITC and Cy3-conjugated secondary antibodies (A0562, A0521, beyotime) for an hour at room temperature, after which they were stained with DAPI (F6057, sigma). Images were captured with fluorescence microscopy (BX63, OLYMPUS). Primary antibodies here included anti-APJ (20341-1-AP, Proteintech), anti-F4/80 (MAB5580-SP, R&D Systems), anti-NOX4 (14347-1-AP, Proteintech), anti-PFKFB3 (A5593, Bimake).
3
Western Blot Analysis of Cellular Proteins
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Equal amounts of proteins from cell lysate or tissues, as determined by BCA protein estimation, were boiled with 5× loading buffer and separated by 10% or 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The proteins were transferred to 0.22 μm polyvinylidene fluoride membranes and then blocked with 5% bovine serum albumin for 1 h at room temperature. Next, the membranes were incubated with primary antibodies, including antibodies against collagen I (1:1000, 14695-1-AP, Proteintech), α-SMA (1:1000, 19245 T, Cell Signaling Technology), PFKFB3 (1:1000, A5593, Bimake), HK2 (1:1000, 66974-1-Ig, Proteintech), β-actin (1:1000, AM1021B, Abcepta, China), LC3B (1:1000, A5202, Proteintech), VDAC1 (1:1000, A5224, Bimake), and Parkin (1:1000, 66674-1-Ig, Proteintech) overnight at 4 °C. The membranes were washed three times with TBS-T buffer and then incubated with secondary antibodies (FDR007, FDM007, Fdbio Science, China) at room temperature for another 1 h. After further washing of the membranes three times with TBS-T buffer, protein signals were detected using a BLT GelView 6000 Pro system.
4
Immunofluorescence Imaging of Cellular Markers
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Paraffin sections (4 µm) and cells were prepared for immunofluorescence and incubated with primary antibody overnight at 4 °C. Then, lung sections and cells were stained with the appropriate secondary antibody and mounted with DAPI (F6057, Sigma Aldrich, USA). Images were captured with a confocal microscope (LSM880, Carl Zeiss) or fluorescence microscope (BX63, OLYMPUS). The primary antibodies used included anti-PFKFB3 (1:200, A5593, Bimake), anti-HK2 (1:100, 66974-1-Ig, Proteintech), anti-Parkin (1:100, 66674-1-Ig, Proteintech), anti-cytochrome c oxidase IV (COX IV; 1:100, 66110-1-Ig, Proteintech), anti-LC3B (1:100, A5202, Bimake/1:200, 83506S, Cell Signaling Technology), anti-LAMP1 (1:100, 32731, Signalway Antibody, USA), and anti-Collagen I (1:100, ab260043, Abcam, USA/1:100, 67288-1-Ig, Proteintech).
5
Histological and Immunohistochemical Analysis of Lung Fibrosis
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For histological analysis, lung tissue sections were subjected to hematoxylin and eosin (H&E) and Masson staining, and the severity of lung fibrosis was analyzed by Ashcroft scoring. For immunohistochemical staining, paraffin-embedded mouse lung sections were incubated with primary antibodies against PFKFB3 (1:200, A5593, Bimake, USA), HK2 (1:200, 66974-1-Ig, Proteintech, USA), and α-SMA (1:200, 19245T, Cell Signaling Technology, USA) according to the manufacturer’s instructions (GK500705, DAKO, Denmark). Images were captured by microscopy (IX73, Olympus or Imager D2, Carl Zeiss).
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