Five-micron thick sections of the TMAs were deparaffinized and rehydrated in sequential xylene and graded ethanol. Antigen retrieval was performed in 10 mM citrate buffer (pH 6.0) in a pressure cooker. Endogenous peroxidase and avidin/biotin were blocked respectively (Vector Laboratories Inc.). Sections were then incubated with 5% normal goat-horse-chicken serum, incubated with primary antibody (Supplemental Table 2 ), incubated with biotinylated secondary antibody (Vector Laboratories Inc.), followed by ABC reagent (Vector Laboratories Inc.), and stable DAB (Invitrogen Corp.). All sections were lightly counterstained with hematoxylin and mounted with Cytoseal XYL (Richard Allan Scientific). Mouse or rabbit IgG were used as negative controls as appropriate.
Stable dab
Manufactured by Thermo Fisher Scientific
Sourced in United States
Stable DAB is a chromogenic substrate used in immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) applications. It produces a brown-colored precipitate at the site of the target antigen or enzyme, allowing for the visualization and detection of the analyte of interest.
Automatically generated - may contain errors
Lab products found in correlation
Biotinylated secondary antibody, by Vector Laboratories (4 mentions)
Abc reagent, by Vector Laboratories (4 mentions)
Cytoseal xyl, by Thermo Fisher Scientific (4 mentions)
Endogenous peroxidase, by Vector Laboratories (3 mentions)
Mouse anti human ifnγ antibody, by BD (2 mentions)
Avidin hrp, by Vector Laboratories (2 mentions)
Biotinylated anti ifn γ antibody, by Mabtech (1 mentions)
Image lab program version 4, by Bio-Rad (1 mentions)
Histostain plus kit, by Zymo Research (1 mentions)
Application suite, by Leica (1 mentions)
Biotinylated anti human ifn γ antibody, by Mabtech (1 mentions)
Rat anti gfap, by Thermo Fisher Scientific (1 mentions)
Rat anti cd45, by Bio-Rad (1 mentions)
Rabbit anti calbindin, by Cell Signaling Technology (1 mentions)
Biotinylated goat anti mouse, by Jackson ImmunoResearch (1 mentions)
Goat anti mouse cy2, by Jackson ImmunoResearch (1 mentions)
Biotinylated goat anti rabbit, by Jackson ImmunoResearch (1 mentions)
Donkey anti rat cy5, by Jackson ImmunoResearch (1 mentions)
Goat anti rabbit cy3, by Jackson ImmunoResearch (1 mentions)
Rabbit anti iba1, by Fujifilm (1 mentions)
9 protocols using stable dab
1
Immunohistochemical Staining of TMAs
2
ELISPOT Assay for Interferon-gamma Detection
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Millipore 96 well filtration plates were pre-treated with 70% EtOH, washed 5x with 1x PBS and then coated with 5 μg/ml mouse-anti-human-IFNγ antibody (BD Pharmingen) over night at 4°C. After blocking with complete RPMI for 2 hours at 37°C, 2x105 PBMCs (prepared as for ICS) were stimulated in triplicates with peptide pools at 1 μg/ml, or PHA (2.5 μg/ml) as positive control, while addition of medium only served as negative control. The peptides and peptide pools used are those described in [43 (link)] and are detailed in S4 Table . Plates were incubated at 37°C for 18–24 hours before washing with cold H2O twice and PBS/T for five times. Biotinylated anti-IFNγ-antibody (Mabtech) was added at 1 μg/ml for 1 hour at 37°C and, after washing, a 1: 2000 dilution of Avidin-HRP (Vector Laboratories) was added, again for 1 hour at 37°C. After final washing, stable DAB (Invitrogen) was added for 2 min, and then the reaction was stopped with water washing. After drying, the numbers of spots in each well were counted with an automated ELISPOT reader (CTL Immunospot).
3
Immunoblotting Analysis of Antibody Reactivity
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Immunoblotting assays were carried out to analyze the specificity and pattern recognition of serum and colostrum antibodies against STAg, as described above [16 (link)]. Briefly, STAg was separated on 12 % SDS-PAGE under non-reducing conditions, and electrotransferred to nitrocellulose membranes. Non-specific interactions were blocked by PBS-T-M5 % incubation, for 2 h at room temperature. Nitrocellulose strips were incubated with serum samples (diluted 1:100 [IgG] or 1:50 [IgM and IgA] in PBS-T plus 1 % of nonfat milk – PBS-T-M1 %) or colostrum (diluted 1:10 [IgG] or 1:5 [IgM and IgA] in PBS-T). Protein-antibody complex was detected by incubation with secondary antibodies, anti-IgG, anti-IgM or anti-IgA labelled with peroxidase (Sigma) for 2 h at room temperature. Reactions were revealed by adding diaminobenzidine (Stable DAB, Invitrogen), and stopped with distilled water. The molecular weight (KDa) for each antigenic fraction was determined using the Image Lab program Version 4.0.1 (Bio-Rad Laboratories, Hercules CA).
4
Immunohistochemistry on Tissue Microarrays
5
Collagen Distribution Analysis in Liver Tissue
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Immunohistochemical analysis for the detection of collagen distribution using light microscopy was performed as previously described25 (link). Briefly, liver tissue samples were fixed in 5% formalin for 12 h, embedded in paraffin and then sliced into 4 μm thick sections. These sections were subsequently deparaffinized with xylene, rehydrated, and pretreated for 30 min at room temperature with PBS blocking buffer containing 10% goat serum. Next, the sections were incubated with primary anti-collagen antibody (Abcam, Cambridge, MA, USA) diluted 1:1,000 in PBS blocking buffer. The antigen-antibody complexes were subsequently visualized with biotinylated secondary antibody (goat anti-rabbit)-conjugated HRP streptavidin (Histostain-Plus Kit, Zymed, South San Francisco, CA, USA) at a dilution of 1:1,500 in PBS blocking buffer. Finally, collagen proteins were detected using a stable DAB (Invitrogen Corp., Carlsbad, CA, USA) and the Leica Application Suite (Leica Microsystems, Switzerland).
6
PBMC IFN-γ ELISPOT Assay
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96 well filtration plates (Millipore) were pre-treated with 70% EtOH, washed five times with 1X phosphate buffered saline and then coated with 5 μg/ml mouse-anti-human-IFNγ antibody (BD Pharmingen) overnight at 4°C. After blocking with complete RPMI for 2 h at 37°C, 2x105 PBMCs were stimulated in triplicates with peptide pools at 1 μg/ml, or phytohaemagglutinin (2.5 μg/ml) as positive control, while addition of medium only served as negative control. The peptides and peptide pools used are those previously described [47 ]. Plates were incubated at 37°C for 18–24 h before washing with cold H2O twice and five times with phosphate buffered saline containing 0.1% (v/v) Tween 20. Biotinylated anti-human IFNγ antibody (cross reactive with IFNγ from non-human primates, Mabtech) was added at 1 μg/ml for 1 h at 37°C and, after washing, a 1: 2000 dilution of Avidin-HRP (Vector Laboratories) was added for 1 h at 37°C. After final washing, stable DAB (Invitrogen) was added to the plate, incubated for 2 min at RT, and then the reaction was stopped by thorough rinsing with water. After drying, the numbers of spots in each well were counted with an automated ELISPOT plate reader (CTL Immunospot).
7
Immunohistochemical Analysis of Bone Metastases
8
Multimodal Analysis of Neuroinflammation
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For immunohistochemistry experiments, tissues were stained with primary antibodies directed to identify microglia (rabbit anti-Iba-1, 1:4000, Wako or mouse anti-Iba-1 kindly provided by Dr. Bruce Trapp), inflammatory monocytes (rat anti-CD45, 1:2000, Bio-Rad), cerebellar neurons (rabbit anti-calbindin, 1:1000, Cell signaling Technology), pro-inflammatory cytokine interleukin-1 beta (rabbit anti-IL1 beta, 1:200, abcam), myelin basic protein (MBP) (rabbit anti-MBP, 1:2000, Invitrogen), and astrocytes (rat anti-GFAP, 1:4000, Invitrogen). Secondary antibodies were purchased from Jackson ImmunoResearch Laboratory as follows: Cy3-goat anti rabbit, Cy5-donkey anti rat, Cy2-goat anti mouse, biotinylated-goat anti rabbit, biotinylated-goat anti mouse, and biotinylated-rabbit anti rat. Biotinylated antibodies are detected by using stable DAB (Thermo Fisher Scientific) containing diaminobenzidine and hydrogen peroxidase.
9
Immunohistochemical Analysis of METTL3
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TMA sections (5 μm) were deparaffinized and rehydrated in xylene and graded ethanol series. Heat-induced antigen retrieval was performed in 10 mM citrate buffer (pH 6.0). Endogenous peroxidase and avidin/biotin were blocked (Vector Laboratories Inc., Burlingame, CA), and TMAs were incubated with 5% normal goat-horse-chicken serum at 4°C overnight. Sections were incubated with the primary antibody recognizing METTL3 (Proteintech, #15073-1-AP, 1:500), followed by the biotinylated secondary antibody (Vector Laboratories Inc.) and ABC reagent (Vector Laboratories Inc.). Staining was detected with stable DAB (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. All sections were counterstained with hematoxylin and mounted with Cytoseal XYL (Richard Allan Scientific, San Diego, CA). Rabbit IgG was used as negative controls.
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