Hearts, kidneys, and lungs from neonatal mice were prepared on postnatal day 1, and hearts, livers, kidneys, and spleens from adult mice were prepared at the age of 11 weeks. Adult mice were euthanized by cervical dislocation, and neonates were decapitated. Organs were excised, rinsed in cold PBS, weighed, snap‐frozen in liquid nitrogen, and stored at −80°C. If organs were used for histological analyses, they were fixed in 4% paraformaldehyde/PBS (Sigma) for 24 to 48 hours. The tissue was subsequently dehydrated through an increasing ethanol series, cleared in toluol, and embedded in paraffin. Five‐micrometer paraffin sections were stained with hematoxylin and eosin (Carl Roth, Karlsruhe, Germany) to assess overall cardiac morphology and tissue composition or with Sirius Red (Direct Red 80, Sigma) to visualize myocardial fibrosis.
Hematoxylin and eosin
Manufactured by Carl Roth
Sourced in Germany
Hematoxylin and eosin are two dyes used together in histology and pathology for the staining of biological tissues. Hematoxylin stains nuclei blue, while eosin stains cytoplasm and extracellular matrix pink.
Automatically generated - may contain errors
Lab products found in correlation
Direct red 80, by Merck Group (2 mentions)
Sp5 laser confocal microscope, by Leica (2 mentions)
Microtome, by Leica (2 mentions)
Paraformaldehyde pbs, by Merck Group (1 mentions)
Optical transparent glass bottom plates, by Greiner (1 mentions)
Af 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Nbp2 24683, by Novus Biologicals (1 mentions)
Entellan, by Merck Group (1 mentions)
Superfrost plus adhesion slides, by Thermo Fisher Scientific (1 mentions)
Axio imager z1 microscope, by Zeiss (1 mentions)
Hematoxylin, by Carl Roth (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Ab20680, by Abcam (1 mentions)
Matrigel, by Corning (1 mentions)
Af555, by Thermo Fisher Scientific (1 mentions)
Ab16669, by Abcam (1 mentions)
Ab21176, by Abcam (1 mentions)
Ab21027, by Abcam (1 mentions)
Sc 6458, by Merck Group (1 mentions)
Sc 6458, by Abcam (1 mentions)
10 protocols using hematoxylin and eosin
1
Organ Preparation and Histological Analysis
2
STING and cGAS Immunohistochemistry and Flow Cytometry
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Tissues were fixed with 4% paraformaldehyde and embedded in paraffin. Sections were stained with hematoxylin and eosin (Carl Roth, Karlsruhe, Germany). STING and cGAS immunohistochemistry was performed on paraffin-embedded tissue sections, using a polyclonal antibody against mouse/human STING raised in rabbit (NBP2-24683, 1:250, Novus Biologicals, Denver, CO, USA or 13657, 1:250, Cell Signaling Technology, Danvers, MA, USA) or against mouse/human cGAS raised in rabbit (201708-T10, 1:125, Sino Biological, Eschborn, Germany). For fluorescent microscopy of STING and cGAS, murine or human preadipocytes were grown on optical transparent glass-bottom plates (Greiner Bio-One GmbH, Frickenhausen, Germany) or glass coverslips and labeled with the same antibodies used for immunohistochemistry, and then visualized with AF488-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA). Histology images were adjusted to equal white balance after acquisition. Flow cytometry analysis was used to detect STING+, cGAS+ macrophages and adipocytes, as described in [24 (link)]. Flow repository identifier of FACS data is FR-FCM-Z236.
3
Lung Tissue Fixation and Histological Processing
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Following intubation of the mice and diaphragm dissection, airways were inflated with 4% (w/v) paraformaldehyde (PFA). Later, the tracheae were knotted, the lungs were excised and transferred into 4% PFA-loaded tubes for tissue fixation overnight at 46 °C. After tissue processing and paraffin embedding, 3 μm thick sections were cut and placed on SuperfrostTM Plus Adhesion Slides (Thermo Scientific). The de-paraffinized sections were stained with hematoxylin and eosin (Carl Roth, Germany) subsequently, and dehydrated respectively in consecutively grading ethanol and xylene solutions (AppliChem, Germany). Dried sections were mounted with entellan (Merck, Germany).
4
Immunohistochemistry of Murine Lungs
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The left lung was extracted, fixed in 4% formaldehyde, and embedded in paraffin. Three‐micrometer lung sections were stained with anti‐mouse GR‐1 or TF antibody, and counterstained with hematoxylin or with hematoxylin and eosin (Carl Roth, Karlsruhe, Germany). Images were obtained with an Axio Imager.Z1 microscope (Carl Zeiss, Vienna, Austria).
5
Teratoma Formation Assay of miPSCs
6
Angiogenesis Assay with Modified Immune Cells
7
Adipocyte Size and Collagen Quantification
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The adipocyte size distribution was determined in formalin-fixed, paraffin-embedded tissues, which were cut into 5 µm sections and stained with hematoxylin and eosin (Carl Roth, Karlsruhe, Germany). Analysis was done with ImageJ using the ADIPOSOFT tool (Schneider et al. 2012 (link)). Sirius Red (Direct Red 80; Sigma, Taufkirchen, Germany) staining was done for 30 min using rehydrated tissues which were afterwards dehydrated as explained before (Haberl et al. 2018 (link)). Sirius Red staining was quantified by ImageJ (Schneider et al. 2012 (link)). Tissue slides were photographed and representative images are shown.
8
Histological Evaluation of Adipose Tissues
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Epididymal WAT, peri-ovarian WAT, and femoral BAT were dissected from 5 to 6 weeks old mice, fixed in 4% para-formaldehyde for 48 h at room temperature, and subjected to dehydration overnight as described previously [35] , [36] (link). Paraffin embedded tissues were sectioned with a microtome (Leica) and mounted on object slides (Carl Roth). Sections were stained with hematoxylin and eosin (Carl Roth) using a multistainer (Leica).
9
Adrenal Gland Histology in Laying Hens
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At the end of the experiment, we excluded the data of one laying hen of the FP group. It showed a constant decrease in body weight until the end of the experiment. Thus, we analyzed seven laying hens kept in SC and six laying hens kept in FP.
The animals were weighed once a month from their 17th week of life until the end of the experiment at their 75th week of life.
Then, all animals were anesthetized with isoflurane and killed by bleeding. Both adrenal glands were extracted and fixed in 4% formaldehyde (Formalin, RotiHistofix, Roth, Karlsruhe, Germany). After 48 h, the adrenal glands were dehydrated with denatured ethanol and xylol (both Roth, Karlsruhe, Germany) and embedded in paraffin wax (Paraplast PLUS, Roth, Karlsruhe, Germany). The fixed organs were cooled at 4 °C until cutting. Subsequently, the adrenal glands were cut into 4 μm thin sections using a microtome (Leica, Wetzlar, Germany). Every 5th section was fixed onto a covered slide with a mixture of protein and glycerin (both Roth, Karlsruhe, Germany) and dried in an incubator at a temperature of 54 °C for an hour afterward. Afterward, the slices were stained with hematoxylin and eosin (Roth, Karlsruhe, Germany).
The animals were weighed once a month from their 17th week of life until the end of the experiment at their 75th week of life.
Then, all animals were anesthetized with isoflurane and killed by bleeding. Both adrenal glands were extracted and fixed in 4% formaldehyde (Formalin, RotiHistofix, Roth, Karlsruhe, Germany). After 48 h, the adrenal glands were dehydrated with denatured ethanol and xylol (both Roth, Karlsruhe, Germany) and embedded in paraffin wax (Paraplast PLUS, Roth, Karlsruhe, Germany). The fixed organs were cooled at 4 °C until cutting. Subsequently, the adrenal glands were cut into 4 μm thin sections using a microtome (Leica, Wetzlar, Germany). Every 5th section was fixed onto a covered slide with a mixture of protein and glycerin (both Roth, Karlsruhe, Germany) and dried in an incubator at a temperature of 54 °C for an hour afterward. Afterward, the slices were stained with hematoxylin and eosin (Roth, Karlsruhe, Germany).
10
Histomorphology of Cornea and Retina post PDT
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Three eyeballs from each group, the PBS control, and the PDT 24hour group (laser treatment performed 24 hours after verteporfin i.v. injection) were excised 1 week after corneal PDT. In addition, three naïve eyes were harvested as normal morphological controls. The excised eyeball was fixed, dehydrated, and paraffin embedded. Afterward, sections were cut at 4 lm and collected on slides. The sections were deparaffinized in xylene, rehydrated in a descending alcohol series to water, and then stained with hematoxylin and eosin (Carl Roth GmbH þ Co. KG, Karlsruhe, Germany), rinsed in water, dehydrated through an ascending series of alcohols and xylenes, and mounted in Neo-Mount (Merck KGaA, Darmstadt, Germany) and coverslipped. The stained sections were examined with an Olympus light microscope (BX53; Olympus). The region of the central cornea and retina were identified in sections, and five sections were imaged in the central cornea and retina region from a section series at 4003 magnification with a digital camera (XM10; Olympus) in each eye. These central cornea images were used to count the keratocytes and corneal endothelial cells manually. The thickness of the retina was measured in the retina region with ImageJ 1.50i (National Institutes of Health, Bethesda, MD, USA).
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