H.E. staining was performed on 5 µm thick paraffin sections. After deparaffinization and rehydration twice in xylene, 99% isopropanol, and decreasing concentrations of ethanol (95%, 80%, 70%) for 12 min each, sections were washed in PBS for 5 min and incubated in Mayer’s Hemalum for 3 min. Excess staining solution was removed by washing with running tap water until the water remained clear. For counterstaining, the sections were incubated in 2% alcoholic eosin solution for 2 min, followed by short dehydration steps in ethanol of increasing concentrations (70%, 80%, 90%), 99% isopropanol, and a final incubation in xylene (2 × 5 min). The slides were mounted in DePeX mounting medium (Serva Electrophoresis GmbH, Heidelberg, Germany). Analysis was performed using an inverted microscope (Observer.Z1, Zeiss).
Depex mounting medium
Manufactured by Serva Electrophoresis
Sourced in Germany
DePeX is a mounting medium used in microscopy. It is a clear, colorless solution that is used to permanently mount specimens on microscope slides. DePeX has a high refractive index, which helps to improve the optical clarity of the mounted specimen.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (5 mentions)
Axiovision 4, by Zeiss (4 mentions)
Axiocam mrc camera, by Zeiss (4 mentions)
Serum block, by Agilent Technologies (4 mentions)
Mayer s hematoxylin, by Merck Group (3 mentions)
Paraformaldehyde (pfa), by Merck Group (3 mentions)
Phosphate buffered saline (pbs), by Merck Group (3 mentions)
Axioskop 2 microscope, by Zeiss (3 mentions)
Dab chromogen, by Thermo Fisher Scientific (3 mentions)
Nuclear fast red solution, by Merck Group (2 mentions)
Glass coverslip, by Carl Roth (2 mentions)
Goat anti rabbit igg hrp secondary antibody, by Agilent Technologies (2 mentions)
Goat anti rabbit igg hrp, by Agilent Technologies (2 mentions)
Alcian blue solution, by Merck Group (1 mentions)
Dab peroxidase substrate kit, by Vector Laboratories (1 mentions)
Mouse anti dcx antibody, by Santa Cruz Biotechnology (1 mentions)
Cresyl violet, by Merck Group (1 mentions)
Axiophot microscope, by Zeiss (1 mentions)
Axio scope a1 fluorescence microscope, by Zeiss (1 mentions)
Bovine serum albumin (bsa), by Carl Roth (1 mentions)
16 protocols using depex mounting medium
1
Hematoxylin and Eosin Staining Protocol
2
Histological Staining for Spinal Cord Imaging
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Consecutive sections of spinal cords were fixed in methanol-acetone (1:1) and stained with hematoxylin and eosin (H&E) in order to provide a reference for spectroscopic imaging. Sections were washed in distilled water and incubated in Meyer’s hematoxylin/hemalum for 3 min. After washing in distilled water, sections were briefly destained in HCl-ethanol and washed using tap water for 5 min. After 3 min of staining in eosin (1% (w/v) eosin G in 80% ethanol), the sections were dehydrated with rising ethanol concentrations, cleared in xylene and coverslipped using DePeX mounting medium (SERVA Electrophoresis GmbH, Heidelberg, Germany).
To provide a reference for spectroscopic imaging of alginate hydrogel implants in spinal cord tissue, sections were stained with alcian blue which is specific for polysaccharides. Alcian blue solution (pH = 2) was placed on sections and the staining success was observed under the microscope. Then, the sections were washed with running tap water for 3 min and stained with nuclear fast red for 10 min. After 10 min of washing with running tap water, the sections were dehydrated with rising ethanol concentrations, cleared in xylene and coverslipped using DePeX mounting medium.
To provide a reference for spectroscopic imaging of alginate hydrogel implants in spinal cord tissue, sections were stained with alcian blue which is specific for polysaccharides. Alcian blue solution (pH = 2) was placed on sections and the staining success was observed under the microscope. Then, the sections were washed with running tap water for 3 min and stained with nuclear fast red for 10 min. After 10 min of washing with running tap water, the sections were dehydrated with rising ethanol concentrations, cleared in xylene and coverslipped using DePeX mounting medium.
3
Mucus Quantification in BCi Cells
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BCi cells polarized in permeable supports were fixed in 4% PFA/PBS for 20 min at room temperature and embedded in paraffin. Paraffin cuts of 5 µm were deparaffinized, stained with standard Alcian blue solution and counterstained with Nuclear Fast Red solution (Sigma-Aldrich, St. Louis, Missouri, USA). After dehydration and clearing steps, sections were mounted in DePeX mounting medium (SERVA Electrophoresis, Heidelberg, Germany). Stains were assessed by light microscopy. Mucus-stained (blue) areas were determined using ImageJ.
4
Immunocytochemistry of CTSS in PDL cells
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PDL cell stimulation with IL-1β (1 ng/mL) and F. nucleatum (OD: 0.025) was performed as described above, and cells were cultured on glass coverslips (Carl Roth, Karlsruhe, Germany) in 24-well plates for 24 h. Cells were prepared for immunocytochemistry by fixation in 4% paraformaldehyde (Sigma-Aldrich, Munich, Germany) at pH 7.4 and room temperature and by permeabilization with 0.1% Triton X-100 (Sigma-Aldrich). Each incubation step was followed by two washing steps with PBS (Sigma-Aldrich). To block unspecific background staining, cells were suspended in serum block (Dako, Hamburg, Germany) for 20 min, followed by incubation with rabbit polyclonal antibody anti-CTSS (Abcam, Cambridge, MA, USA; 1 : 250) at 4°C overnight. Cells were then labeled with a goat anti-rabbit IgG-HRP secondary antibody (Dako) for 45 min. Antibody binding was made visible by DAB chromogen (Thermo Fisher Scientific, Waltham, MA, USA) staining for 10 min at room temperature. Counterstaining with Mayer's hematoxylin (Merck, Darmstadt, Germany) for 1 min was followed, and finally, coverslips were mounted with DePex mounting medium (SERVA Electrophoresis, Heidelberg, Germany). An Axioskop 2 microscope (Carl Zeiss, Jena, Germany) with an AxioCam MRc camera (Carl Zeiss) and the AxioVision 4.7 software (Carl Zeiss) were used for standardized imaging.
5
Immunohistochemical Localization of NTN-1 and NEO-1 in VAT
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Immunohistochemistry was performed as previously described to evaluate the cellular localization of NTN-1 and NEO-1 in VAT [27 (link)]. Briefly, VAT sections (6 µm) from OB-NG and OB-T2D patients were formalin-fixed, dewaxed in xylene and rehydrated in decreasing concentrations of ethanol. Sections were incubated overnight at 4 °C with a goat anti-human NTN-1 monoclonal antibody (R&D Systems, Minneapolis, USA) diluted 1:100 in Tris buffered saline (TBS, Merck) and a rabbit anti-human NEO-1 monoclonal antibody (R&D Systems) diluted 1:50 on TBS. After rinsing the slides with TBS, they were incubated for 1 h at room temperature with anti-goat and anti-rabbit secondary antibodies conjugated with DakoTM RealTM EnVisionTM horseradish peroxidase (DakoCytomation, Glostrup, Denmark). Sections were dehydrated in increasing concentrations of ethanol, mounted with DePeX mounting medium (Serva, Heidelberg, Germany) and observed under a Zeiss Axiovert CFL light microscope (Zeiss, Göttingen, Germany) at 20X. A negative control slide was included in which the primary antibody was replaced by TBS to assess nonspecific staining.
6
Immunostaining of DCX+ Neurons
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One series of free-flowing brain sections was used for immunostaining. Sections were washed three times in phosphate buffered saline (PBS), pH 7.4, to remove the cryoprotective solution. Endogenous peroxidase was then blocked by PBS with 0.05% Triton X-100 (PBST), 10% H2O2 and 1% methyl alcohol for 30 min. The sections were washed three times in PBST and then incubated in 10% NGS and 0.1% BSA for 2 h. Next, the mouse anti-DCX antibody (1:500, Santa Cruz Biotechnology, Heidelberg, Germany) was applied overnight at 4 °C. The following day, sections were washed and incubated for 1.5 h in biotin-conjugated secondary polyclonal anti-rabbit antibody (1:300, Vector Laboratories, Burlingame, CA, USA). Sections were washed three times in PBST and incubated in PBS with StreptAvidine (1:400, Vector Laboratories, Burlingame, CA, USA). The color reaction was induced with horseradish peroxidase substrate, 3,3′-diaminobenzidine chromium and 0.05% H2O2 (DAB Peroxidase Substrate Kit, Vector Laboratories, Burlingame, CA, USA). The sections were washed three times in phosphate buffer and then placed on gelatinized slides, dehydrated and coversliped with DePeX mounting medium (Serva, Electrophoresis GmbH, Heidelberg, Germany).
7
Immunocytochemical Analysis of SSTR2 in PDL Fibroblasts
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PDL fibroblasts were cultured on glass coverslips in the presence and absence of IL-1β, F. nucleatum, leptin or visfatin (Carl Roth, Karlsruhe, Germany) for 24 h. Afterwards, cells were fixed in 4% paraformaldehyde (Sigma-Aldrich, Munich, Germany) at pH 7.4 and room temperature for 10 min, washed with PBS (Sigma-Aldrich), and treated with 0.1% Triton X-100 (Sigma-Aldrich) for 5 min. Background staining was prevented by the application of serum block for 20 min (Dako, Hamburg Germany). After washing, cells were incubated with a rabbit polyclonal anti-SSTR2 antibody (10 μg/ml; R&D Systems) at 4 °C overnight. Goat anti-rabbit IgG-HRP (Dako) was applied as a secondary antibody for 45 min. Antibody binding was visualized by DAB chromogen (Thermo Fisher Scientific, Waltham, MA, USA) staining for 10 min at room temperature. Two washing steps with PBS (Sigma-Aldrich) were always performed between the incubation steps. Finally, cells were counterstained with Mayer’s Hematoxylin (Merck Eurolab, Dietikon, Switzerland) for 1 min and coverslipped with DePex mounting medium (Serva Electrophoresis, Heidelberg, Germany). Standardized photomicrographs were taken with an Axioskop 2 microscope (Carl Zeiss, Jena, Germany) equipped with an AxioCam MRc camera (Carl Zeiss) and the AxioVision 4.7 software (Carl Zeiss).
8
Cresyl Violet Staining for Histology
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Three to six random sections from different regions were used per SC analyzed. Sections were washed twice in PBS and then permeabilized using a 50% alcohol/water solution for 30 min. Then, sections were washed in deionized distilled water for 3 min and submerged in an activated Cresyl Violet (Merck) filtered solution for 30 min. Sections, then, were washed in distilled water for 3 min and decolored in 70% alcohol/water for a minimum time of 30 min. When white matter was decolored, sections were submerged three times in 100% alcohol for 5 min and then in isoparaffin H twice for 5 min. Finally, sections were allowed to dry for 3 min and mounted in DePeX mounting medium (Serva).
9
H&E Staining of Human Brain Tissue
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Several adjacent sections of human GBM and HBC brain tissue were stained with haematoxylin and eosin (H&E), to provide greater morphological and histopathological detail. Slides were air dried for 20 min at room temperature before incubation with haematoxylin solution (Sigma; Haematoxylin Solution, Harris Modified; HHS128) for 5 min. Slides were washed in running tap water until water ran clear, incubated in Scott’s Tap Water (NaHCO₃, 3.5 g/L; MgSO₄, 20 g/L in distilled water) for 1 min and then washed in running tap water a second time before incubating with eosin solution (Sigma; Eosin Y Solution, Aqueous; HT110280) for 10 min. A final wash was performed in running tap water until water ran clear. Slides were sequentially dehydrated (2 min at each step) through an alcohol series (30, 70, and 100% ethanol), cleared with xylene for 1 h before mounting with DePeX mounting medium (Serva) and being covered with a cover slip. Slides were left to cure overnight.
10
Immunohistochemical Analysis of Mouse Skin and Organotypic Cultures
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4 μM sections on polylysine coated slides from formalin fixed, paraffin-embedded organotypic cultures and mouse skin were analysed. Sections were deparaffinised by washing in 100 % Xylene, rehydrated through washing in decreasing concentrations of ethanol. Sections were then incubated in 3 % hydrogen peroxide in methanol for 20 min to inhibit endogenous peroxidises. Antigen unmasking was performed by boiling the tissue sections in 10 mM citric buffer for 3 min in a beaker in a microwave followed by 15 min resting at RT. Sections were then blocked in 50 % horse serum in PBS (v/v) for 30 min. Primary antibody was diluted in 2 % horse serum/PBS and incubated overnight at 4 °C. A biotinylated secondary antibody was applied and slides were visualized using a streptavidin-biotin-peroxidase detection system (Vectastain ABC or M.O.M. kit, Linaris, Dossenheim, Germany) using DAB (3,3’-diaminobenzidine) liquid substrate (Biogenex, Fremont, CA, USA). Sections were counterstained in Gills Haematoxylin and dehydrated through washing in increasing concentrations of ethanol, then mounted in DePeX mounting medium (Serva, Heidelberg, Germany) and visualised using an Zeiss Axiophot microscope and imaging software.
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