For hematoxylin and eosin (H&E) staining, tissues were fixed with buffered 10% formalin solution (SF93-20; Fisher Scientific, Fair Lawn, NJ, USA) overnight at 4°C. Dehydration was achieved by immersion in a graded ethanol series, i.e., 70%, 80%, 95%, and 100% ethanol for 40 min each. Tissues were embedded in paraffin and subsequently cut into ultra-thin slices (5 μm) using a microtome. The sections were deparaffinized by xylene (Fisher Scientific) and rehydrated by decreasing concentrations of ethanol and PBS. Tissue sections were then stained with H&E and slides were scanned with an Aperio ScanScope. For frozen sections, tissues were fixed with periodate-lysine-paraformaldehyde (PLP) and dehydrated with 30% sucrose in PBS at 4°C overnight. The sections were incubated with anti-F4/80 (Santa Cruz, no. sc-25830), anti-Gr-1 (BioLegend, no. 108404), anti-CD11b (BioLegend, no. 101204), anti-EpCAM (Abcam, ab71916) and anti-angiotensin converting enzyme-2 (ACE-2) (ProSci, no. 3217) at a 1:100 dilution at 4°C overnight. The signal was visualized with the secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen), and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). The slides were scanned using an Aperio ScanScope or visualized with a confocal laser scanning microscope (Nikon, Melville, NY, USA) as described.44 (link)
Sf93 20
Manufactured by Thermo Fisher Scientific
Sourced in United States
The SF93-20 is a laboratory centrifuge designed for general-purpose applications. It has a maximum speed of 6,000 rpm and can accommodate a rotor with a capacity of 20 tubes. The centrifuge provides reliable and consistent separation performance for a variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Xylene, by Thermo Fisher Scientific (10 mentions)
Scanscope, by Thermo Fisher Scientific (3 mentions)
Anti cd11b, by BioLegend (2 mentions)
Digital camera, by Nikon (2 mentions)
Oct compound, by Thermo Fisher Scientific (2 mentions)
Software, by Nikon (2 mentions)
Confocal laser scanning microscopy, by Nikon (2 mentions)
Anti epcam, by Abcam (1 mentions)
Ace 2, by ProSci (1 mentions)
Anti gr1, by BioLegend (1 mentions)
Anti f4 80, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Laser scanning confocal microscope, by Nikon (1 mentions)
Vcam 1, by Cell Signaling Technology (1 mentions)
Isolectin b4, by Thermo Fisher Scientific (1 mentions)
Podocin, by Merck Group (1 mentions)
Masson s trichrome, by Wuhan Servicebio Technology (1 mentions)
Sirius red, by Wuhan Servicebio Technology (1 mentions)
Q color 5 digital camera, by Olympus (1 mentions)
18 protocols using sf93 20
1
Comprehensive Tissue Preparation and Staining
2
Cardiac Tissue Histological Analysis
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Some heart tissues were fixed with neutral‐buffered 10% formalin solution (SF93–20; Fisher Scientific, Pittsburgh, PA). The other tissues were embedded in frozen OCT compound (4585; Fisher Health Care, Houston, TX). The paraffin and frozen sections were prepared with 10 µm in thickness. We prepared all of the tissues under the same conditions. Paraffin sections were stained with Masson’s trichrome staining and hematoxylin–eosin. Some frozen sections were stained with dihydroethidium staining for reactive oxygen species (ROS) measurement. Some frozen sections were immune stained with VCAM‐1 (1:150; Cell Signaling, Danvers, MA). Isolectin B4 (frozen section) was performed to measure ECs (1:150; Invitrogen, Carlsbad, CA) and capillary density. Images were obtained with a Nikon microscope, Nikon digital camera, and Nikon software (Nikon, Tokyo, Japan). Seven random microscopic fields were chosen for analysis with image analysis software (Image J, National Institutes of Health).
3
Tissue Sectioning and Staining Protocol
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Serial sections were put in neutral-buffered 10% formalin solution (SF93–20; Fisher Scientific, Pittsburgh, PA, USA). Other sections were put in frozen OCT compound (4585; Fisher Health Care, Houston, TX, USA). In the same conditions, we cut these tissues to 10 µm in thickness. Paraffin parts were prepared for Masson’s trichrome staining and hematoxylin and eosin (H&E) staining. Some frozen sections were prepared for reactive oxygen species (ROS) measurement using DHE staining. Images were obtained using Nikon digital camera and analyzed in Nikon software (Nikon, Tokyo, Japan). Four to six random parts were picked for measurement using Image J (NIH, Bethesda, MD, USA).
4
Histological Analysis of Tissue Lipids
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Live tissues were fixed with buffered 10% formalin solution (SF93– 20; Fisher Scientific, Pittsburgh, PA), embedded in frozen optimal-cutting-temperature compound, and 10μm frozen sections prepared. Some sections were stained with Hematoxylin & eosin (H & E), and some sections were stained with Oil Red O working solution for lipid analysis [25 (link), 26 (link)]. Masson trichrome staining was also performed on adjacent sections to measure the degree of fibrosis. The area percentage of lipid or fibrosis was quantified by measuring 6 random microscopic fields using image-analysis software (Image J, NIH).
5
Histopathological Analysis of Kidney Tissue
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Kidney tissue specimens underwent fixation with 10% formalin (SF93-20; Fisher Scientific, Pittsburgh, PA, USA). Histological features were assessed by hematoxylin and eosin (H&E; Servicebio, Wuhan, China) and Periodic Acid Schiff (PAS; Servicebio) staining. Fibrosis was assessed by the ratio of fibrotic area to total area detected by Masson’s trichrome (Servicebio) and Sirius red (Servicebio) staining. Kidney apoptosis was examined by TUNEL (Servicebio). Kidney specimens underwent embedding in frozen optimal cutting temperature compound (Fisher HealthCare, Houston, TX, USA) and sectioning at 8 µm for immunostaining. The specimens underwent incubation with primary antibodies targeting SIRT1 (1:100; Abcam, Cambridge, MA, USA) and podocin (1:100; Sigma, Shanghai, China). This was followed by incubation with second antibodies conjugated with fluorescein isothiocyanate (1:500). For quantitation, 10 random high-power fields in mouse kidney samples were assessed with Image J (NIH, Bethesda, MD, USA).
6
Renal Cortex Histological and Immunohistochemical Analysis
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The renal cortex tissues were fixed with buffered 10% formalin solution (SF93–20; Fisher Scientific, Pittsburgh, PA, USA), embedded in frozen OCT compound (4583; Sakura Finetek, Torrance, CA, USA) and 10 μm frozen sections prepared. Some sections were stained with haematoxylin & eosin. Some were directly immunostained with Alexa 488‐labelled Isolectin B4 (IB4) for ECs, smooth muscle actin (SMA) and a NG2 antibody for pericytes (1:100; Abcam). Other sections were immunostained with Notch3, PHD2, FSP‐1 and TGF‐β1 primary antibodies (1:200) followed by incubation with second antibodies conjugated with fluorescein isothiocyanate (FITC) or Cy3 (1:500). Photomicrographs were obtained with an Olympus BX51 microscope, a Q‐Color5 digital camera and a Q‐Capture Suite acquisition software (Olympus, Tokyo, Japan). The area percentage of fluorescence intensity was quantified at six random microscopic fields using image analysis software (Image J, NIH, Bethesda, MD, USA). Masson trichrome staining was also performed to measure the degree of fibrosis (blue) in the renal cortex.
7
Tissue Fixation and H&E Staining
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Tissues were fixed in buffered 10% formalin solution (SF93‐20; Fisher Scientific, Fair Lawn, NJ) overnight at 4°C for haematoxylin and eosin (H&E) staining. Dehydration was achieved by immersing the tissue cassette for 40 min in a graded ethanol series of 70%, 80%, 95% and 100% ethanol. Tissues were fixed in paraffin and then sliced into ultrathin (5 μm) slices with a microtome. Tissue sections were deparaffinised in xylene (Fisher), rehydrated in increasing concentrations of ethanol in PBS, stained with H&E, and scanned with an Aperio ScanScope (IL, USA).
8
Tissue Immunofluorescence Staining Protocol
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Tissues were fixed with buffered 10% formalin solution (SF93-20; Fisher Scientific, Fair Lawn, NJ) overnight at 4 °C and dehydrated by immersing in a graded ethanol series, 70%, 80%, 95%, 100% ethanol for 40 min each. Tissues were embedded in paraffin and subsequently cut into ultra-thin slices (5 μm) using a microtome. Tissue sections were deparaffinized by xylene (Fisher) and rehydrated by decreasing concentrations of ethanol and PBS, stained with hematoxylin and eosin (H&E) and scanned with an Aperio ScanScope. For tissue immunofluorescent staining, slides were washed three times (5 min each) with PBST (PBS, 0.1% Tween 20). The tissue was permeabilized by incubating the slides in 1% Triton X-100 in PBS at 25 °C for 15 min and then washed three times in PBST. After blocking for 1 h at 25 °C in blocking buffer (PBS containing 10% bovine serum albumin (BSA)), slides were incubated overnight in a humidity chamber with anti-F4/80 polyclonal antibody (Thermo Fisher). Antibodies were diluted 1:50 in blocking buffer. Following three PBST washes, slides were incubated with Alexa 488-conjugated secondary antibody at a 1:500 dilution (Invitrogen). Slides were then washed and nuclei counterstained with DAPI.
9
Brain Tissue Histological Processing
10
Tissue Preparation for H&E Staining
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For hematoxylin and eosin (H&E) staining, tissues were fixed with buffered 10% formalin solution (SF93-20; Fisher Scientific, Fair Lawn, NJ) overnight at 4 °C. Dehydration was achieved by sequential immersion in a graded ethanol series of 70%, 80%, 95%, and 100% ethanol for 40 min each. Tissues were embedded in paraffin and subsequently cut into ultrathin slices (5 μm) using a microtome. Tissue sections were deparaffinized in xylene (Fisher), rehydrated in decreasing concentrations of ethanol in PBS, stained with H&E, and the slides were scanned with an Aperio ScanScope using a method previously described 23 .
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