Human lungs were maintained in 10% formalin for 24 hours and then embedded in paraffin. Paraffin-embedded lungs were cut into 4 μm sections. After removing the paraffin, endogenous peroxidases were inactivated in an aqueous solution containing 3% H2O2 and 10% methanol and antigen retrieval was performed heating the samples in citrate buffer (10mM citric acid, pH 6.0). The sections were then blocked in bovine serum albumin (5%), incubated with anti-ACE2 antibody (R&D Systems cat. n° AF933, dilution 1:100) and with biotinylated secondary antibody against goat IgGs (Vector Laboratories cat. n° BA-9500, dilution 1:250). Proteins were visualized using the ABC Peroxidase Standard Staining Kit (ThermoFisher) followed by 3,3′-Diaminobenzidine (DAB) Enhanced Liquid Substrate System (Sigma Aldrich). Counterstaining was done with hematoxylin.
Abc peroxidase standard staining kit
Manufactured by Thermo Fisher Scientific
The ABC Peroxidase Standard Staining Kit is a laboratory reagent designed for use in immunohistochemistry applications. The kit provides a standardized method for the detection and visualization of target proteins or antigens in biological samples. It contains the necessary components to perform the avidin-biotin-peroxidase complex (ABC) staining technique.
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Lab products found in correlation
Phospho histone h2a x ser139 antibody, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3 asp175 antibody, by Cell Signaling Technology (1 mentions)
Dab peroxidase hrp substrate kit, by Vector Laboratories (1 mentions)
Goat serum, by Merck Group (1 mentions)
Haematoxylin, by Thermo Fisher Scientific (1 mentions)
3 3 diaminobenzidine (dab), by Abcam (1 mentions)
Aperio at2 system, by Leica (1 mentions)
Anti gr 1 antibody, by BioLegend (1 mentions)
Imagequant tl, by GE Healthcare (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Ab64238, by Abcam (1 mentions)
Ab220210, by Abcam (1 mentions)
Vector red substrate kit, by Vector Laboratories (1 mentions)
Dp71 digital imaging system, by Olympus (1 mentions)
3 30 diaminobenzidine, by Vector Laboratories (1 mentions)
Immpact sg peroxidase substrate, by Vector Laboratories (1 mentions)
Avidin biotin solution, by Vector Laboratories (1 mentions)
Anti nedd4 antibody, by Abcam (1 mentions)
Dpx mountant, by Merck Group (1 mentions)
Biotinylated goat anti rabbit secondary antibody, by Vector Laboratories (1 mentions)
9 protocols using abc peroxidase standard staining kit
1
Immunohistochemical Detection of ACE2
2
Immunohistochemical Analysis of DNA Damage and Apoptosis
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The tumor samples were paraffin-embedded and then sliced into 5 μm thicknesses. The sections were deparaffinized, rehydrated, incubated with phospho-histone H2A.X (Ser139) antibody (1:400 dilution, #9718, Cell Signaling Technology) or cleaved caspase-3 (Asp175) antibody (#9661, Cell Signaling Technology), and then stained by an ABC peroxidase standard staining kit (Thermo Fisher Scientific) containing biotinylated affinity-purified goat anti-rabbit IgG (1:1000 dilution, 32054, Thermo Fisher Scientific) and a DAB peroxidase (HRP) substrate kit (Vector Laboratories) according to the manufacturer’s protocol. Finally, the sections were observed under a microscope Olympus BX51 (Olympus) with three different fields taken for each group.
3
Immunohistochemical Analysis of Paraffin-Embedded Tissues
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Paraffin-embedded tissue samples were cut into 5 µm sections, deparaffinized and rehydrated. For antigen retrieval, slides were boiled in citrate buffer (0.01 M, pH 6.0) using a microwave oven on high power for 5 min and cooled down to room temperature. After incubation in 3% aqueous H2O2 to quench the endogenous peroxidase, the sections were washed in PBST (PBS with 0.1% Tween-20, v/v) washing buffer, blocked with 5% goat serum (EMD Millipore) diluted in PBST at room temperature for 1 h and incubated with primary antibodies diluted in 2% goat serum in PBST at 4 °C overnight.
For immunohistochemistry, slides were incubated with appropriate biotinylated secondary antibodies (Supplementary Table3 ) for 1 h, and then processed according to the ABC Peroxidase Standard Staining Kit (Thermo Fisher Scientific) for 30 min. The slides were stained with 3,3′-diaminobenzidine (Abcam) for 5 s to 5 min and counterstained with haematoxylin (Thermo Fisher Scientific) for 45 s. The images were scanned using the Leica Aperio AT2 system at Stanford Human Pathology/Histology Service Center. Serial sections were incubated with GS and MYC-tag primary antibodies, and a cluster of cells (at least 20 cells) positive both for GS and MYC-tag were counted as early foci. The antibodies used in this study are shown in Supplementary Table 3 .
For immunohistochemistry, slides were incubated with appropriate biotinylated secondary antibodies (Supplementary Table
4
Neutrophil Infiltration Quantification
5
Western Blot and Dot Blot Analyses
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For western blot analysis, samples were loaded onto 10% or 12% polyacrylamide gels.
Where indicated, AviCD9LELAvi, as well as a CD9LEL constructs that were not tagged with the Avi sequence were produced and 6 μL of each recombinant peptide was incubated with SA (Nanovex Biotechnologies) for 15 min at RT using two different ratios (3-fold molar excess and 10-fold molar excess of SA/tetraspanin LEL). Samples were boiled in Laemmli buffer and resolved in a polyacrylamide gel. After electrophoresis, the proteins were transferred to a PVDF membrane (Biorad).
For dot blot analysis, samples were spotted onto a 0.22 μm pore size nitrocellulose membrane (GE Healthcare) and let to dry.
Membranes were subsequently blocked with 5% non-fat dry milk and blotted with ABC Peroxidase Standard Staining Kit (Thermo Fisher Scientific) for 30 min at RT or incubated overnight with primary antibodies at 4°C in a humidified chamber. After washing, membranes were probed with secondary antibody anti-mouse horseradish peroxidase (Thermo Fisher Scientific) and Enhanced chemiluminescent (ECL) HRP substrate (Thermo Scientific). Chemiluminescence signal was detected using the LAS4000 mini Image System analyser from Fujifilm and software ImageQuant-TL (GE Healthcare).
Where indicated, AviCD9LELAvi, as well as a CD9LEL constructs that were not tagged with the Avi sequence were produced and 6 μL of each recombinant peptide was incubated with SA (Nanovex Biotechnologies) for 15 min at RT using two different ratios (3-fold molar excess and 10-fold molar excess of SA/tetraspanin LEL). Samples were boiled in Laemmli buffer and resolved in a polyacrylamide gel. After electrophoresis, the proteins were transferred to a PVDF membrane (Biorad).
For dot blot analysis, samples were spotted onto a 0.22 μm pore size nitrocellulose membrane (GE Healthcare) and let to dry.
Membranes were subsequently blocked with 5% non-fat dry milk and blotted with ABC Peroxidase Standard Staining Kit (Thermo Fisher Scientific) for 30 min at RT or incubated overnight with primary antibodies at 4°C in a humidified chamber. After washing, membranes were probed with secondary antibody anti-mouse horseradish peroxidase (Thermo Fisher Scientific) and Enhanced chemiluminescent (ECL) HRP substrate (Thermo Scientific). Chemiluminescence signal was detected using the LAS4000 mini Image System analyser from Fujifilm and software ImageQuant-TL (GE Healthcare).
6
Immunohistochemical Analysis of TNF-alpha in Kidney
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The fixed left and right kidneys were processed and embedded in paraffin. The tissues were cut at 5 μm thickness and mounted on the glass slides. The sections were deparaffinized, rehydrated, and antigen unmasked with 10 mM sodium citrate (pH 6.0) using microwave technique. The sections were blocked with 10% normal horse serum (ab7484, Abcam) in PBS for 1 h and then incubated overnight with an anti-TNF alpha antibody (mouse monoclonal, ab220210, 1:200, Abcam). After washing, the sections were incubated with 0.3% H2O2 for 15 min and with a goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, biotin (1: 5,000, Invitrogen) for 1 h. For detection, the sections were washed and further incubated with ABC peroxidase standard staining kit (Thermo Scientific™) for 30 min and with a DAB substrate kit (ab64238, Abcam) for 5 min. The sections were counter stained with hematoxylin solution modified acc. to Gill III (Sigma-Aldrich) for a few seconds. All slides were dehydrated and mounted with mounting medium. The images were capture in an imaging system (Nikon, Tokyo, Japan).
7
Immunohistochemical Evaluation of Kidney Biomarkers
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A series of immunohistochemical (IHC) examinations with appropriate antibodies were used to examine biomarkers or histological changes in kidneys. An ED-1 antibody was used for macrophages, and an MDA antibody was used for lipid peroxidation. Nrf2 and DNA repair enzyme 8-oxoguanine DNA glycosylase 1 (OGG1) were detected with their specific antibodies. Immunohistochemical staining was performed according to the standard avidin-biotin-peroxidase complex method with an ABC Peroxidase Standard Staining Kit (cat. 32020; Thermo Fisher Scientific, Inc.). Oxidative damaged DNA was detected by an 8-OHdG antibody and immunohistochemical staining was performed with a VECTASTAIN ABC-AP kit (cat. AK-5002; Vector Laboratories, San Francisco, CA, USA) and a Vector Red Substrate kit (cat. SK-5100; Vector Laboratories). The information for the antibodies is listed in Supplementary Table S2 .
8
Immunohistochemical Analysis of Xenograft Tissues
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Athymic balb/c nude mice were euthanized and tissues were harvested and fixed in formalin for the preparation of paraffin sections. Paraffin-embedded tissue sections were deparaffinized in xylene, 95, 90, and 70% ethanol, followed by phosphate buffered saline (PBS). Epitopes were unmasked with 20 mg/ml proteinase K in PBS with 0.1% Triton X-100. Sections were stained with hematoxylin and eosin (H & E) or immunostained overnight at 4°C with the Notch2 (1:100), E-cadherin (1:100), N-cadherin (1:100), vimentin antibody (1:100) and IL-6 antibody (1:100). After washing in PBS, a 1:200 dilution of biotinylated goat anti-rabbit IgG or anti-mouse IgG antibody in a blocking solution was applied to the sections and incubated for 30 min. After washing in PBS, the ABC reagent (ABC Peroxidase Standard Staining Kit-Thermo Fisher Scientific) was applied to the sections and incubated for 30 min. After washing in PBS, color reaction was performed with 3, 30-diaminobenzidine (Vector Laboratories) and the slides were washed with PBS. After counter-staining with hematoxylin and clearing with a graded ethanol series and xylene, the sections were mounted with Canada balsam. Observations and photography were conducted using a microscope (Olympus) equipped with a DP71 digital imaging system (Olympus).
9
NEDD4 Immunohistochemistry in Parkinson's Disease
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Postmortem human SNpc paraffin-embedded sections from sporadic PD patients and control individuals were obtained from the Neurological Tissue Bank (Biobank-HC-IDIBAPS) and stained as previously reported [6 (link)]. Sections were dewaxed with xylene and rehydrated by incubation in ethanol series (100%, 95%, 70% and 50%). Antigen retrieval was performed with Tris-EDTA buffer (10 mM Tris-base pH = 9, 1 mM EDTA, 0.05% Tween 20) in a vegetable steamer at 100°C for 20 minutes. Then, slides were blocked in superblock-PBS for 2 hours at room temperature and were incubated with avidin/biotin solution (Vector Laboratories, Burlingame, CA). Sections were incubated O/N at 4°C with anti-NEDD4 antibody (Abcam), washed with TBS-0.025% Triton X-100 and incubated for 15 minutes with 0.3% H2O2 in TBS. Secondary antibody incubation was performed for 2 hours at room temperature with biotinylated goat anti-rabbit secondary antibody (Vector Laboratories). Finally, slides were incubated with ABC Peroxidase Standard Staining Kit (Thermo Fisher Scientific) for 30 minutes and with ImmPACT SG Peroxidase Substrate (Vector Laboratories) for 20 minutes. Sections were washed, dehydrated by incubation in ethanol series (50%, 70%, 95% and 100%) and xylene, mounted with DPX mountant (Sigma-Aldrich) and examined under the microscope.
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