Histological slides were obtained from the Department of Anatomic Pathology at Bellaria Hospital, University of Bologna (Italy). For immunostaining of paraffin-embedded sections with SOD2 (Millipore 06-984), samples were deparaffinized in xylene and rehydrated in water, and antigen retrieval was performed in citrate buffer. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 minutes. All washes were performed using 50 mM Tris-HCl, pH 7.6, three times for 2 minutes. Blocking solution was applied for 30 minutes prior to the incubation with SOD2 antibody at a dilution of 1/100 for 2 hours at room temperature. Secondary antibody was incubated for 1 hour and the sections developed using the LSAB+ kit peroxidase (DAKO, Cat#K0690) for 10 minutes. Sections were then counterstained in haematoxylin and mounted with Permount (Fischer Scientific, Cat#SP15-100).
Lsab kit peroxidase
Manufactured by Agilent Technologies
Sourced in Denmark
The LSAB+ kit peroxidase is a laboratory equipment product designed for immunohistochemical staining procedures. It is used to detect the presence of specific target proteins in tissue samples. The kit includes the necessary reagents and components required for the staining process.
Automatically generated - may contain errors
Lab products found in correlation
Permount, by Thermo Fisher Scientific (2 mentions)
Anti nitrotyrosine antibody, by Thermo Fisher Scientific (2 mentions)
Anti cox 2 antibody, by Abcam (1 mentions)
Anti tnf α antibody, by Abcam (1 mentions)
Anti inos, by Abcam (1 mentions)
Nitrotyrosine, by Merck Group (1 mentions)
Diaminobenzidine dab, by Agilent Technologies (1 mentions)
Image pro plus, by Media Cybernetics (1 mentions)
Anti ki67 antibody, by Abcam (1 mentions)
Anti vimentin, by R&D Systems (1 mentions)
Microscope slide, by Thermo Fisher Scientific (1 mentions)
Anti plasminogen, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Eclipse ti inverted microscope, by Nikon (1 mentions)
Dab chromogen kit, by Biocare Medical (1 mentions)
12 protocols using lsab kit peroxidase
1
Immunohistochemical SOD2 Detection
2
Immunohistochemical Analysis of Inflammatory Markers
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Five sections (5 μm in thickness) from each group were cut and adhered to slides treated with 3-aminopropyltriethoxysilane (APES (Sigma, USA)). Briefly, sections were deparaffinized with xylene and rehydrated in graded ethanol (100 to 70%). To minimize endogenous peroxidase activity, the slides were treated with 10% (v/v) H2O2 in water for fifteen minutes. The sections were washed with 0.01 M PBS (pH 7.2) and then blocked with 1% BSA, 0.2% Tween 20 in PBS for 1 h at room temperature. The sections were incubated overnight at 4°C with anti-TNF-α antibody (ABCAM, CA, USA, 1 : 250), anti-IL-1β antibody (GenWay, San Diego, CA, USA, 1 : 250), anti-COX-2 antibody (ABCAM, CA, USA, 1 : 400), and anti-iNOS (ABCAM, CA, USA, 1 : 50). The antigen-antibody reaction was visualized with avidin-biotin peroxidase (Dako Universal LSAB + Kit, Peroxidase), using 3,3-diaminobenzidine as the chromogen. The slides were counterstained with hematoxylin. Positive staining resulted in a brown reaction product. Five pictures at the same magnification were quantitatively analyzed using the Gimp 2.6 software program (GNU Image Manipulation Program, UNIX platforms).
3
Immunohistochemical Analysis of SOD2 Expression
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Histological slides were obtained from the Department of Anatomic Pathology at Bellaria Hospital, University of Bologna (Italy). For immunostaining of paraffin-embedded sections with SOD2 (Millipore 06-984), samples were deparaffinized in xylene and rehydrated in water, and antigen retrieval was performed in citrate buffer. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 min. All washes were performed using 50 mM Tris-HCl, pH 7.6, three times for 2 min. Blocking solution was applied for 30 min before the incubation with SOD2 antibody at a dilution of 1/100 for 2 h at room temperature. Secondary antibody was incubated for 1 h and the sections developed using the LSAB+ kit peroxidase (DAKO, Glostrup, Denmark; catalogue number K0690) for 10 min. Sections were then counterstained in hematoxylin and mounted with Permount (Fischer Scientific, Waltham, MA, USA; catalogue number SP15-100).
4
Immunohistochemical Analysis of Tumor Tissue
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For immunohistochemical analysis, tumor tissue samples were fixed in 10% buffered
formalin and embedded in paraffin. Blocks were sectioned at 4 µm, deparaffinized, and
rehydrated. The streptavidin-biotin method (LSAB kit + peroxidase; Dako®,
Denmark) was used to detect p53 protein (anti-p53 monoclonal antibody, clone DO-7,
1:100 dilution; Dako®, catalog No. M7001) and dopamine D2receptor (anti-DRD2 polyclonal antibody, 1:150 dilution; Chemicon®, USA,
catalog No. AB5084P). Normal human pituitary and breast tissues were used as positive
controls for dopamine D2 receptor and p53 protein, respectively (19 (link),20 (link)). As
a negative control, the primary antibodies were replaced with saline.
The criterion for positive intracellular dopamine D2 receptor and p53
protein expression was the presence of at least one group of cells with clearly
marked cytoplasm or nuclei, respectively (5 (link),20 (link)). Specimens were analyzed by
two independent observers, using light microscopy.
formalin and embedded in paraffin. Blocks were sectioned at 4 µm, deparaffinized, and
rehydrated. The streptavidin-biotin method (LSAB kit + peroxidase; Dako®,
Denmark) was used to detect p53 protein (anti-p53 monoclonal antibody, clone DO-7,
1:100 dilution; Dako®, catalog No. M7001) and dopamine D2receptor (anti-DRD2 polyclonal antibody, 1:150 dilution; Chemicon®, USA,
catalog No. AB5084P). Normal human pituitary and breast tissues were used as positive
controls for dopamine D2 receptor and p53 protein, respectively (19 (link),20 (link)). As
a negative control, the primary antibodies were replaced with saline.
The criterion for positive intracellular dopamine D2 receptor and p53
protein expression was the presence of at least one group of cells with clearly
marked cytoplasm or nuclei, respectively (5 (link),20 (link)). Specimens were analyzed by
two independent observers, using light microscopy.
5
Immunohistochemical Staining Protocol
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Immunohistochemistry was performed as previously described [59] (link). RCA sections were incubated with avidin–biotin two-step blocking solution (Vector SP-2001) to inhibit background staining and 3% hydrogen peroxide to inhibit endogenous peroxidase. Non-serum protein block (Dako X909) was then applied to inhibit non-specific protein binding. Sections were incubated at 4°C overnight with primary antibodies KCNN4 (1∶600, Chemicon), nitrotyrosine (1∶400, Chemicon), or SMαA (1∶200, DAKO). After appropriate washing steps, sections were incubated with biotinylated secondary antibody in phosphate-buffered saline containing 15 mM sodium azide and peroxidase-labeled streptavidin (Dako LSAB+ kit, peroxidase, K0690). Diaminobenzidine (DAB, Dako) was applied 5 minutes for visualization of the reaction product, sections were then counterstained with haematoxylin, dehydrated, and coverslipped. Images of the sections were obtained using an Olympus BX40 photomicroscope and Spot Insight Color camera (Diagnostic Instruments). The relative area and mean density of positive staining for KCNN4 were determined for each section of interest utilizing ImagePro Plus (Media Cybernetics).
6
Immunohistochemical Analysis of Tumor Markers
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Sections of 4 lm were cut from the paraffin-embedded tumor samples, dewaxed with xylene, gradually rehydrated and then stained with hematoxylin and eosin (H&E), according to standard procedure. Immunohistochemistry reagents were purchased from Dako, Denmark. Endogenous peroxidase activity in the sections was blocked by incubation with the dual endogenous enzyme block for 30 min. All studied sections were boiled in target retrieval solution (pH 9) for 20 min. After cooling and washing twice for 5 min with washing buffer, the histology and immunohistochemistry (IHC) reactions were performed using rabbit polyclonal anti-Ki 67 antibodies diluted 1:200 (Abcam, UK), antimouse AC133 epitope of Prominin-1 (CD133; Santa Cruz, USA) and anti-vimentin (R&D, USA). Tested sections were incubated with antibodies accordingly to the manufacturers' instructions. The test antigen was visualized using biotinylated antibodies, the streptavidin-biotinylated peroxidase complex (Universal Dako LSAB ? kit, Peroxidase, Code K0679) and diaminobenzidine. All sections were shortly afterwards counterstained with Meyer's hematoxylin. The test procedure included a negative control in which the specific antibody was substituted by PBS.
7
Histological Analysis of Tissue Necrosis and DNA Damage
8
Histological Analysis of Liver Tissue
9
Quantifying HSP 70 in Earthworms
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HSP 70 in extruded coelomocytes of treated E. fetida were sampled and determined by western blot on day 28 after exposure to spiked natural soil. The immune-peroxidase staining protocol was used on methanol-fixed cytospin preparations after blocking of endogenous peroxidase with 3% H2O2 as described previously [20 (link)]. Monoclonal mouse anti-human anti-HSP 70 were diluted 1:1000 with 0.25% Triton X in phosphate buffer supplemented with 1% BSA (overnight, 4°C) followed by the labeled streptavidin biotin method (Dako LSAB peroxidase kit).
10
Histopathological Assessment of Liver Necrosis
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