Hematoxylin eosin

Organs for histopathological analysis were fixed in 4% formaldehyde (Genta Medical, UK) followed by paraffin embedding and sectioning. Sections were stained with Hematoxylin-Eosin (Merck) for microscopic examination. FACS-purified erythroid precursors were transferred onto slides using a cytospin centrifuge and stained with May-Grünwald and Giemsa solutions (Merck). Morphological examination was performed using AxioImager Z2 Upright Wide-field Microscope (Zeiss).

Before being killed, all animals received 1 h HFS followed by 1 h interval in their home-cage. Subsequently, the rats were anesthetized with Nembutal (75 mg kg⁻¹) and perfused transcardially with 4% paraformaldehyde fixative solution. The brains were cryoprotected by overnight 15% sucrose treatment and frozen with CO₂. The brains were cut serially (10 series) on a cryostat (MICROM, Walldorf, Germany) into 30 μm coronal sections and stored at −80 °C until stainings were performed. A standard hematoxylin–eosin (Merck, Darmstadt, Germany) staining was performed to examine the localization of the electrode tips and screen for histological damage. We performed immunohistochemical stainings for c-Fos and 5-HT on the basis of previously established methods.^{38 (link), 39 (link)} For the staining methods, microscopic analysis and quantification, please refer to the Supplementary Methods.

The heart tissue samples (6 rat hearts in each group) were fixed with 4% paraformaldehyde and then embedded in paraffin. The slides were stained with hematoxylin–eosin (Merck, Darmstadt, Germany) following standard procedures. Photomicrographs of the heart sections were obtained in at least 6 separate fields × 2 slides × 3 left ventricle regions per condition (6 rat hearts in each group) for data quantification.

At 2 dpi (4 dpf) the xenografted embryos were fixed in 4% paraformaldehyde for 1 h at room temperature, followed by paraffin embedding for hematoxylin & eosin staining or OCT embedding for fluorescent immunohistochemistry staining. Embryos were respectively sectioned with microtome or cryostat, along the sagittal plane at a thickness of 8 μm.
Histopathological analysis was performed on paraffin sections stained by hematoxylin & eosin (Merck KGaA, Darmstadt, Germany) and digitally imaged using Nikon Eclipse E600 microscope.
Cryostat sections were incubated with FITC anti-IgG Primary Antibody (Roche, Basel, Switzerland) and Hoechst 33342 counterstained. Digital images of the stained sections were generated using a Nikon A1 confocal microscope. Pyknotic cells was counted at 60 X magnification within the epifluorescence DAPI image.

PBECs cultured on filter membranes were fixed with 3.7% paraformaldehyde at room temperature for 15 minutes. Afterwards, cells were embedded in paraffin, thin sections were cut and stained with hematoxylin-eosin (Merck, Germany). Light microscopy was performed using a Biorevo BZ-9000 microscope (Keyence, Germany).

Smears of freshly taken swab samples were fixed with M-Fix^TM spray fixative (Merck, Darmstadt, Germany) according to the manufacturer's instructions. Cells were stained with Hematoxylin & Eosin (Merck, Darmstadt, Germany) or with Wright-Giemsa stain (Sigma-Aldrich, St.Louis, USA). Epithelial cells and leukocytes could easily be discerned by their morphology. For each sample we analyzed 50 randomly taken microscopic fields (corresponding to 328 ± 144 cells; cell counting was performed independent of pyrosequencing results).

Chloroform, diethyl ether, methanol, ethanol (80%), ethyl acetate, ferric sulfate, lead acetate, Dil. HNO_3, sodium hydroxide, disodium citrate, glacial acetic acid, formic acid, methyl cellulose, isopropanol, xylene, paraffin wax, Hematoxylin-Eosin, and metformin hydrochloride were purchased from ACS, Merck. 10% formalin, and potassium ferrocyanide were purchased from BDH, Ltd. (England). Sulfuric acid was purchased from Farm Italia Carrloerba (Italy). Standard rutin trihydrate from Fluka, no.78095. Standard quercetin from Sigma no. Q4954. Standard gallic acid from Fluka no.91215. Streptozotocin (Sigma Chemical Company, USA) was used to induce diabetes in rats. All other chemicals are purchased from Merk with 99% purity. All chemicals were of analytical grade.