For the histological evaluation, kidney tissues were extracted, sectioned, and stained with standard protocols. Masson’s trichrome staining was performed with Masson Trichrome staining (Sigma-Aldrich, St. Louis, MO, USA) kits following the manufacturers’ instruction. The primary antibody used for immunostaining was anti-alpha smooth muscle actin (SMA) (NBP1-30894, Novus Biologicals, Littleton, CO, USA) immunohistochemical staining was performed with Envision DAB + kits (Dako, Basel, Switzerland) following the manufacturer’s instructions.
Nbp1 30 894
Manufactured by Novus Biologicals
Sourced in United States
NBP1-30,894 is a laboratory product offered by Novus Biologicals. It is a specific reagent or component used in scientific research or laboratory procedures. No further details about its core function or intended use can be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Masson trichrome staining, by Merck Group (1 mentions)
Ix81 inverted fluorescence microscope, by Olympus (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Ab6671, by Abcam (1 mentions)
Ab59396, by Abcam (1 mentions)
Ab78494, by Abcam (1 mentions)
Ab64548, by Abcam (1 mentions)
Ab21685, by Abcam (1 mentions)
Sc 13063, by Santa Cruz Biotechnology (1 mentions)
Nb600 408, by Novus Biologicals (1 mentions)
Sc 7219, by Santa Cruz Biotechnology (1 mentions)
Apoptag kit, by Merck Group (1 mentions)
Ab46545, by Abcam (1 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
4 protocols using nbp1 30 894
1
Kidney Histological Evaluation
2
Immunofluorescent Staining of Smooth Muscle Markers
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The cell layer was washed with phosphate buffer solution (PBS, Sigma-Aldrich, USA) and fixed with 4% paraformaldehyde (Sigma-Aldrich, USA) for 30 min at room temperature. After three washes with PBS, the cells were permeabilized with 0.25% Triton X-100 for 20 min. Non-specific sites were blocked with 3% bovine serum albumin (Sigma Aldrich, USA) for 30 min. The cells were incubated with a primary antibody overnight at 4 °C. Antibodies used were α-SMA (1:200, NBP1-30,894, Novus Biologicals, USA), calponin (1:200, 13,938–1-AP, Proteintech, USA), and smoothelin (1:200, orb158429, Biorbyt, UK). After three washes in PBS, α-SMA, calponin, and smoothelin were incubated with goat anti-rabbit antibodies; Dylight488 and Dylight596, respectively. The nuclei were stained with DAPI. Images were captured using an Olympus IX-81 inverted fluorescence microscope (Olympus IX-81, Olympus Corporation, Japan).
3
Histological Analysis of Kidney Injury
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Tissue sections were stained with hematoxylin and eosin (HE), Periodic acid-Schiff’s (PAS) and Masson’s trichrome stains using standard protocols. The mesangial area was determined from the PAS stained sections and the fibrotic area from the Masson’s trichrome stained sections as previously described28 (link). The antibodies used in this study were those against FXR (sc-13063, Santa Cruz), Grp78 (ab21685, Abcam), Chop (ab59396, Abcam), 4-hydroxynonenal (4-HNE, ab46545, Abcam), Kim-1 (ab78494, Abcam), 8-oxo-2′-deoxyguanosine (8-oxo-dG, ab64548, Abcam), SDHA (#11998, CellSignaling, Danvers, MA, USA), tumor necrosis factor (TNF, ab6671, Abcam), CD4 (sc-7219, Santa Cruz, Dallas, TX, USA), aSMA (NBP1-30894, Novus Biologicals, Littleton, CO, USA) and Collagen I (ColI, NB600-408, Novus Biologicals). TUNEL staining on kidney paraffin sections was performed with an ApopTag kit (Millipore, Billerica, MA, USA), according to the manufacturer’s instructions.
4
Western Blot Analysis of Myofibroblast Markers
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After 5 days of culture, the cells were collected and washed three times with pre-cooled PBS. The cells were lysed using RIPA lysis buffer (Beyotime Biotechnology, China) and protease inhibitors (Thermo Fisher, USA) on ice for 30 min. The lysate was centrifuged at 12,000 rpm and 4 ℃ for 15 min, and the supernatant was collected. The protein concentration in each sample was determined using a BCA assay. Protein samples were separated using 10% SDS-PAGE gels with 15 μg per well, electro-transferred to nitrocellulose membranes, and blocked with milk. The membranes were then incubated with α-SMA (1:2000, Rabbit polyclonal, NBP1-30,894, Novusbio, USA), CNN 1 (1:2000, Rabbit polyclonal, 13,938–1-AP, Proteintech, USA), and anti-histone 3 (1:2000, Rabbit polyclonal, 4499 s, Cell signaling, USA). The protein samples were incubated with goat anti-rabbit conjugated horseradish peroxidase (1:20,000; Abbkine, China). Blots were developed using an enhanced chemiluminescence method according to the manufacturer’s protocol (Biosharp, China).
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