Adipose and liver tissues were excised and fixed in PBS buffered 10% formalin for 2 days. Following paraffin embedding, the tissue sections were stained with hematoxylin and eosin (H&E) and Masson's trichrome using standard protocols. For immunohistochemistry, sections were deparaffinized. After antigen retrieval and blockage of endogenous peroxidase, sections were stained with primary antibodies against endotrophin22 (link)(1:1,000, non-purified), F4/80 (0.1 mg ml—1, 1:100; Santa Cruz, CA) and Mac-2 (1 mg ml—1, 1:1,000; Tebu-Bio, France) followed by biotinylated secondary antibodies (0.85 mg ml–1, 1:1,000 anti-rat; 0.5 mg ml–1, 1:1,000 anti-mouse and 1.1 mg ml—1, 1:1,000 anti-rabbit, respectively) (Dako, Glostrup, Denmark). Secondary antibodies were detected using DAB chromogen A kit (Dako) following the company's protocol. The slides were also counterstained with hematoxylin. All the images were acquired with the Coolscope microscope (Nikon, Japan). Quantification of adipocyte diameters was done on H&E stained sections by the ImageJ software from the NIH (http://rsbweb.nih.gov/ij/download.html ). Quantification of the blue colour density for the Masson's trichrome staining was also done by ImageJ software.
Coolscope microscope
Manufactured by Nikon
Sourced in Japan
The Coolscope is a compact and portable microscope designed for laboratory use. It features high-resolution optics and a user-friendly interface for efficient sample observation and analysis. The Coolscope is a versatile instrument suitable for a range of laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Biotinylated secondary antibody, by Agilent Technologies (1 mentions)
F4 80, by Santa Cruz Biotechnology (1 mentions)
Mac 2, by Tebu-Bio (1 mentions)
Dab chromogen a kit, by Agilent Technologies (1 mentions)
Cxcr4, by Merck Group (1 mentions)
Normal goat serum (ngs), by Merck Group (1 mentions)
Cxcl12, by Abcam (1 mentions)
Stat3, by Santa Cruz Biotechnology (1 mentions)
5 protocols using coolscope microscope
1
Adipose and Liver Tissue Histology
2
Histological Analysis of Liver and Kidney
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A piece of fresh liver and a piece of kidney were fixed in formaldehyde and embedded in paraffin. The 4 μm-thick sections were stained with hematoxylin and eosin (H&E) or Masson's Trichrome staining. The slides were examined under a Coolscope microscope (Nikon). For transmission electron microscopy analyses, small pieces of the liver were immediately fixed in 2% glutaraldehyde at 4 °C. The sample was dehydrated in a grade series of ethanol and embedded in an epoxy resin. Tissue was surveyed with a series of 70 nm sections and observed with a Jeol 1400JEM transmission electron microscope equipped with a Orius 100 camera and digital micrograph.
3
Histological Evaluation of Liver Tissue
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A piece of fresh liver (of the largest lobe) was fixed in 10% formalin for 48 h, followed embedding in paraffin. Sections (4-μm-thick) were stained with haematoxylin and eosin (H&E) or Masson's trichrome by the “CiQLe” platform (University Lyon 1). The slides were examined under a Coolscope microscope (Nikon).
4
Immunohistochemical Analysis of OSA Markers
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IHC was performed on 4--fixed paraffin-embedded tissue derived from 12 OSA (9 males and 3 females with mean age 7.7 years, range 0.5-14). As previously described [55] ,
deparaffinized/rehydrated sections were incubated in citrate-antigen retrieval buffer, nonspecific immunoreactivity was blocked with 10% normal goat serum (Sigma-Aldrich) and primary antibodies applied overnight at 4 °C. The following antibodies were used: Oct4 (Abcam), CXCR4 (Sigma-Aldrich), CXCL12
(Abcam), CD117 and STAT3 (SantaCruz). Real Envision Detection System Peroxidase/DAB+, mouse/rabbit (Dako) was used for the detection according to the manufacturer's instructions. Counterstaining with hematoxylin was performed. For each staining, a negative control, was included without the primary antibody or with rabbit/mouse IgG. Images were captured using a Nikon Coolscope microscope. The intensity of immunoreaction and the percentage of positive cells were evaluated, and a score ranging from 0 , independently by two pathologists (A.R. and C.C.).
deparaffinized/rehydrated sections were incubated in citrate-antigen retrieval buffer, nonspecific immunoreactivity was blocked with 10% normal goat serum (Sigma-Aldrich) and primary antibodies applied overnight at 4 °C. The following antibodies were used: Oct4 (Abcam), CXCR4 (Sigma-Aldrich), CXCL12
(Abcam), CD117 and STAT3 (SantaCruz). Real Envision Detection System Peroxidase/DAB+, mouse/rabbit (Dako) was used for the detection according to the manufacturer's instructions. Counterstaining with hematoxylin was performed. For each staining, a negative control, was included without the primary antibody or with rabbit/mouse IgG. Images were captured using a Nikon Coolscope microscope. The intensity of immunoreaction and the percentage of positive cells were evaluated, and a score ranging from 0 , independently by two pathologists (A.R. and C.C.).
5
Histological Analysis of Adipose Tissue
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Tissues were collected and fixed in 4% formalin (in PBS) overnight, embedded in paraffin and sectioned at a thickness of 5 μm. Slides were dewaxed with xylene, rehydrated with descending grades of ethanol and then rinsed with distilled water. Tissue sections were then stained with hematoxylin and eosin. ImageJ was used for quantification of adipocyte size and area from histological slides. In brief, images were transformed into 8-bit and the image threshold was set to selectively visualize adipocyte prior analyses. All the images were acquired with the Coolscope Microscope (Nikon).
For immunohistochemistry, tissue sections were deparaffinized, antigen was retrieved by steaming for 30 min and then cooled to room temperature. Slides were blocked with 5% BSA for 1 h, incubated with anti-BrdU primary antibody (rat monoclonal, Abcam) overnight at 4°C and secondary antibody conjugated with cyanine 5 (eBioscience). Images were observed with a confocal laserscanning microscope (FV1000, Olympus MicroImaging).
For immunohistochemistry, tissue sections were deparaffinized, antigen was retrieved by steaming for 30 min and then cooled to room temperature. Slides were blocked with 5% BSA for 1 h, incubated with anti-BrdU primary antibody (rat monoclonal, Abcam) overnight at 4°C and secondary antibody conjugated with cyanine 5 (eBioscience). Images were observed with a confocal laserscanning microscope (FV1000, Olympus MicroImaging).
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