Paraffin embedded lung sections were subjected to antigen retrieval in boiling citrate buffer and stained with polyclonal goat anti-influenza NP antibody or goat IgG isotype control (Abcam), followed by biotin-conjugated donkey anti-goat secondary antibody (Jackson Immunoresearch). Staining was developed using the Vectastain ABC kit and DAB substrate (Vector Laboratories) as per manufacturer’s instructions. The extent of bronchiolar NP staining was semi-quantitatively analyzed by a blinded investigator, using the following scoring system. The mean of all airways in one section (≥10) was reported for each mouse.0, No NP staining. 1, 1-25 % of epithelial cells in airway stained. 2, 26-50 % of epithelial cells in airway stained. 3, 51-75 % of epithelial cells in airway stained. 4, 51-75 % of epithelial cells in airway stained. An additional +1 score was added where one or more patches of dense staining was observed, giving a maximum score of 5 per bronchiole.
Goat igg isotype control
Manufactured by Abcam
The Goat IgG isotype control is a laboratory reagent used as a control in immunoassays. It serves as a reference point to help distinguish specific from non-specific binding of antibodies in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Dab substrate, by Vector Laboratories (2 mentions)
Goat anti influenza np antibody, by Abcam (2 mentions)
Biotin conjugated donkey anti goat secondary antibody, by Jackson ImmunoResearch (2 mentions)
Vectastain abc kit, by Vector Laboratories (2 mentions)
Ifn α and ifn β, by PBL Assay Science (1 mentions)
Ifn γ, by R&D Systems (1 mentions)
Ifn α, by Abcam (1 mentions)
Ifn α, by PBL Assay Science (1 mentions)
4 protocols using goat igg isotype control
1
Semi-Quantitative Analysis of Influenza NP in Lung Sections
2
Semi-Quantitative Analysis of Influenza NP in Lung Sections
3
Blocking IFN-λ4 Signaling in HepG2 Cells
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The stable HepG2-ISRE-Luc cells were seeded in 96-well plates that were either untransfected or transfected with corresponding constructs. After 24 h, the cells were treated for 2 h with the following antibodies: 20 μg/mL of a blocking α-IL10R2 antibody (R&D Systems), 40 μg/mL of a goat isotype IgG control (Abcam), a range (20, 10, 5, 2.5, or 1.25 μg/mL) of a custom rabbit monoclonal α-IFN-λ4 antibody (Prokunina-Olsson and others 2013 (link)), or 40 μg/mL of a rabbit isotype IgG control (Cell Signaling). Recombinant human interferons—10 ng/mL of custom IFN-λ3 (Prokunina-Olsson and others 2013 (link)), IFN-α and IFN-β (PBL Assay Science), or IFN-γ (R&D Systems) were added to the untransfected cells pretreated with the antibodies. Negative controls included nontreated cells, phosphate-buffered saline (PBS) in media, and mock transfection with control-Halo constructs. The cells were assayed for luciferase expression of the ISRE-Luc reporter 48 h post-transfection. All transfections and treatments were done in 8 biological replicates. For other experiments, inducible stable HepG2 cells were seeded into 96-well plates and protein expression was induced for 12 h. Antibodies (20 μg/mL of rabbit monoclonal α-IFN-λ4 or 40 μg/mL of rabbit IgG control) were added to shared culture media for 48 h.
4
Investigating JAK/STAT Signaling in IFN-λ3 and IFN-α Responses
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The stable HepG2-ISRE-Luc cells were transfected with corresponding constructs in 96-well plates; untransfected cells were treated with human recombinant interferons-IFN-α (2 ng/mL; PBL Assay Science) or custom IFN-λ3 (10 ng/mL). All experiments were represented by 8 biological replicates. The media was replaced 24 h post-transfection by 100 μL of full culture media with 1 or 10 μM JAK inhibitor [active against JAK1, JAK2, and JAK3, #420097; EMD Millipore, in 0.1% dimethyl sulfoxide (DMSO)]. For IL10R2 blocking experiment, the transfection culture media was replaced by 100 μL of media with 20 μg/mL of an α-IL10R2 blocking antibody (MAB874; R&D Systems) or 20 μg/mL of a goat isotype IgG control (Abcam).
For treatment experiments, cells were pretreated for 1 h with the JAK inhibitor or for 2 h with the α-IL10R2 or IgG control antibodies and then treated with IFN-λ3 (10 ng/mL) or IFN-α (2 ng/mL). Negative controls included untreated cells, cells treated with 0.1% DMSO in media, and cells transfected with control-Halo construct. The cells were assayed for ISRE-Luc reporter 48 h post-transfection, which corresponds to 24 h post-treatment with JAK inhibitor and antibodies.
For treatment experiments, cells were pretreated for 1 h with the JAK inhibitor or for 2 h with the α-IL10R2 or IgG control antibodies and then treated with IFN-λ3 (10 ng/mL) or IFN-α (2 ng/mL). Negative controls included untreated cells, cells treated with 0.1% DMSO in media, and cells transfected with control-Halo construct. The cells were assayed for ISRE-Luc reporter 48 h post-transfection, which corresponds to 24 h post-treatment with JAK inhibitor and antibodies.
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