Kidney tissues were fixed in 10% buffered formalin solution for 30 min at room temperature and dehydrated in 75% ethanol overnight, followed by paraffin embedding. Serial sections (4 µm, n=3/group randomly selected) were stained with hematoxylin for 15 min and eosin for 5 min at temperature to assess pathological changes using a BX40 upright light microscope (Olympus Corporation). Kidney damage scores were determined according to the extent of kidney injury, as previously described (13 (link),14 (link)), by two blinded researchers. Scoring was primarily based on the presence or absence of hemorrhaging, tubular cell necrosis, tubular dilatation and cytoplasmic vacuole formation. The grading system was scored as follows: 0, 0% damage (normal kidney); 1, 0-5% damage (minimal damage); 2, 5-25% damage (mild damage); 3, 25-75% damage (moderate damage); and 4, 75-100% damage (severe damage).
Bx40 upright light microscope
Manufactured by Olympus
Sourced in Japan
The BX40 is an upright light microscope manufactured by Olympus. It is designed for routine observation and analysis of a wide range of samples. The microscope features high-quality optics and a sturdy, ergonomic design to provide reliable performance in a laboratory setting.
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Lab products found in correlation
Histone simple stain kit, by Nichirei Biosciences (2 mentions)
Rabbit anti cd68 antibody, by Abcam (1 mentions)
Histofine simple stain kit, by Nichirei Biosciences (1 mentions)
Rabbit anti lc3 antibody, by Proteintech (1 mentions)
Leica rm2235, by Leica camera (1 mentions)
Leica cm1850uv, by Leica camera (1 mentions)
Rabbit anti p62 antibody, by Proteintech (1 mentions)
Rabbit anti nrf2 antibody, by Proteintech (1 mentions)
Rabbit anti nox4 antibody, by Proteintech (1 mentions)
Rabbit anti ho 1 antibody, by Solarbio (1 mentions)
Rabbit anti collagen 3 antibody, by Solarbio (1 mentions)
Collagen 3, by Proteintech (1 mentions)
Sp 9001 splink detection kits, by OriGene (1 mentions)
Splink detection kit, by OriGene (1 mentions)
10 protocols using bx40 upright light microscope
1
Histological Assessment of Kidney Damage
2
Renal Histopathological Analysis
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Renal tissues were fixed for 48 hours with 4% paraformaldehyde, dehydrated, embedded in paraffin, and cut into 4‐μm slices, before being stained with periodic acid‐Schiff (PAS) and Masson's trichrome to assess mesangial matrix expansion and collagen deposition, respectively. More than 25 glomeruli per group were counted, and the mean was used for analysis. All sections were evaluated under an Olympus (Tokyo, Japan) BX40 upright light microscope.
3
Histological Analysis of Heart Tissue
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Hearts were dissected free from the surrounding connective tissue, and fixed with 4% paraformaldehyde, embedded in paraffin, and then cut into slices using a microtome (Leica RM 2235 or Leica CM1850UV; Leica, Solms, Germany). The slices were then mounted onto glass slides, and histological examinations were performed. Immunohistochemistry was performed using Histofine Simple Stain kit (Nichirei, Tokyo, Japan), according to the manufacturer’s instructions. Briefly, sections were deparaffinised with xylene and then rehydrated in a descending ethanol series. Sections were treated with 3% H2O2 in methanol for 15 min to inactivate endogenous peroxidases and then incubated with a primary antibody against p62 (rabbit anti-p62 antibody, 1:200; Proteintech); LC3 (rabbit anti-LC3 antibody, 1:200; Proteintech); CD68 (rabbit anti-CD68 antibody, 1:250; Abcam) at room temperature for 1 h. All sections were examined under an Olympus BX40 upright light microscope (Olympus, Tokyo, Japan).
4
Histological Analysis of Cardiac Tissue
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Cardiac samples were fixed in 10% buffered formalin solution and embedded in paraffin. Paraffin-embedded cardiac tissue slices were deparaffinized via immersion in xylene (3 times, 5 min each), then rehydrated in a descending alcohol series. Then, serial sections (4 µm) were stained with H&E and examined microscopically using a BX40 upright light microscope (Olympus, Tokyo, Japan).
5
Immunohistochemical Analysis of Cellular Markers
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Immunohistochemical analysis was performed using the HistoneSimple stain kit (Nichirei, Tokyo, Japan), according to the manufacturer’s instructions. Paraffin-embedded sections were deparaffinised with xylene and then rehydrated in a descending series of ethanol washes. The sections were treated for 15 min with 3% H2O2 in methanol to inactivate endogenous peroxidases and then incubated at 4 °C overnight with primary antibodies to SIRT1 (rabbit anti-SIRT1 antibody, 1:300; Solarbio), NRF2 (rabbit anti-NRF2 antibody, 1:200; Proteintech), NOX4 (rabbit anti-NOX4 antibody, 1:200; Proteintech), TGF-β (rabbit anti-TGF-β antibody, 1:200; Solarbio), HO-1 (rabbit anti-HO-1 antibody, 1:100; Solarbio), collagen III (rabbit anti-collagen III antibody, 1:100; Solarbio), Smad3 (rabbit anti-Smad3 antibody, 1:100; Solarbio), Bax (rabbit anti-Bax antibody, 1:50; Solarbio), Bak (rabbit anti-Bak antibody, 1:100; Solarbio), Bcl-2 (rabbit anti-Bcl-2 antibody, 1:50; Solarbio) and Bcl-xl (rabbit anti-Bcl-xl antibody, 1:100; Solarbio). All sections were examined microscopically using a BX40 upright lightmicroscope (Olympus, Tokyo, Japan).
6
Cardiac Tissue Analysis in Mice
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Mice were anesthetized with isoflurane, and hearts were fixed by perfusion with 10% buffered formalin. Hearts were fixed overnight at room temperature, transferred into 70% ethanol, and then embedded in paraffin. Paraffin-embedded cardiac tissue slices were deparaffinised via immersion in xylene (3 times, 5 min each), then rehydrated in a descending alcohol series (100, 90, 80, and 70% alcohol, 5 min each). Histology changes was detected by staining sections with Masson’s trichrome, WGA, and PAS staining. Images were acquired microscopically using a BX40 upright lightmicroscope (Olympus, Tokyo, Japan). WGA staining was used to measure the cross-sectional area of cardiomyocytes.
7
Immunohistochemical Analysis of Renal Fibrosis Markers
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Kidney tissues were fixed in 10% buffered formalin solution for 30 min at room temperature and dehydrated in 75% ethanol overnight, followed by paraffin embedding. Immunohistochemical analysis was performed using the SP-9001 SPlink Detection kits (OriGene Technologies, Inc.), according to the manufacturers' instructions. Paraffin-embedded sections (4 µm) were deparaffinized with xylene and subsequently rehydrated in a descending series of ethanol washes (100, 90, 85 and 75%; 5 min each). The sections were treated for 15 min at 98°C with 3% H2O2 in methanol to inactivate the endogenous peroxidase activity and were subsequently incubated at room temperature for 1 h with primary antibodies rabbit against collagen I (cat. no. 14695-1-AP; 1:200; ProteinTech Group, Inc.), collagen III (cat. no. 22734-1-AP; 1:200; ProteinTech Group, Inc.), LC3 (cat. no. 14600-1-AP; 1:200; ProteinTech Group, Inc.) and p62 (cat. no. 55274-1-AP; 1:200; ProteinTech Group, Inc.). The secondary antibody from SPlink Detection kits (OriGene Technologies, Inc.) was incubated with the tissue for 30 min at room temperature. All sections were examined using a BX40 upright light microscope (Olympus BX43; Olympus Corporation). For each staining, 3×7 sections (6 mice) per group were analyzed and the representative images were presented. All image analyses were performed by a blinded reviewer.
8
Kidney Tissue Processing and Fibrosis Analysis
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Kidney tissues from each group were at room temperature stored in 10% formalin, dehydrated in an ascending series of alcohol (75, 85, 90 and 100%; 5 min each) and finally embedded in paraffin wax. Then, 4 µm-thick paraffin sections were sliced from these paraffin-embedded tissue blocks. The tissue sections were subsequently deparaffinized via immersion in xylene (3 times; 5 min each) and rehydrated using a descending series of alcohol (100, 90, 85 and 75%; 5 min each). Biopsy samples were stained at room temperature using Masson's trichrome stain to investigate changes in the kidney morphology and fibrosis. Blue staining represented collagen accumulation. All sections were examined using a BX40 upright light microscope (Olympus BX43; Olympus Corporation).
9
Cardiac Tissue Histological Analysis
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Cardiac tissues were fixed in 10% buffered formalin solution for 30 minutes and
dehydrated in 75% ethanol overnight, followed by paraffin embedding. Serial
sections (4 µm, n = 3/group) were stained with H&E, and the lesion area in
the cardiac tissue was observed using a BX40 upright light microscope (Olympus,
Tokyo, Japan).
dehydrated in 75% ethanol overnight, followed by paraffin embedding. Serial
sections (4 µm, n = 3/group) were stained with H&E, and the lesion area in
the cardiac tissue was observed using a BX40 upright light microscope (Olympus,
Tokyo, Japan).
10
Immunohistochemical Analysis of Hippocampal GFAP in Mice
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The brains of all mice were perfusion-fixed with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4) following a heparinized saline flush. The brains were dehydrated and embedded in paraffin. Serial 7 μm coronal sections were cut using a microtome. Paraffin sections of the hippocampus were used for the immunohistochemical analysis, which was performed using the Histone Simple stain kit (Nichirei, Tokyo, Japan) according to the manufacturer's instructions. Paraffin-embedded sections were deparaffinized with xylene and then rehydrated in a descending series of ethanol washes. The sections were treated for 15 min with 3% H2O2 in methanol to inactivate endogenous peroxidases and then incubated at 4°C overnight with a primary antibody against glial fibrillary acidic protein (GFAP; rabbit anti-GFAP, 1 : 500; Z0334, Dako, Carpinteria, CA, USA). All sections were examined microscopically using a BX40 upright light microscope (Olympus, Tokyo, Japan).
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