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.
Biotin conjugated donkey anti goat secondary antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States
Biotin-conjugated donkey anti-goat secondary antibody is a laboratory reagent used to detect the presence of goat primary antibodies in various immunoassay techniques. It is a secondary antibody that binds to the primary goat antibody and is conjugated with biotin, a small molecule that can be used for signal amplification or detection purposes.
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4 protocols using biotin conjugated donkey anti goat secondary antibody
1
Semi-Quantitative Analysis of Influenza NP in Lung Sections
2
Immunohistochemical Staining Procedure
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Immunohistochemical staining was performed according to standard procedures using the same antibody described above and a biotin-conjugated donkey anti-goat secondary antibody (Jackson Laboratories, West Grove, PA). Sections were developed with diaminobenzidine (DAB) staining and were counterstained with hematoxylin. The staining intensity and ranges were determined by experienced pathologists.
3
Semi-Quantitative Analysis of Influenza NP in Lung Sections
4
Immunohistochemical Analysis of GPNMB in Colorectal Cancer
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Clinical samples for IHC were obtained from CRC patients who received treatment at Gifu University (Gifu, Japan) from January 2010 to January 2016. Seven sets of naive primary tumors and remaining liver metastatic tumors after chemotherapy regimens, such as folinic acid, fluorouracil, and oxaliplatin (FOLFOX) containing anti-EGFR therapy, were used for this analysis 2 (link),3 (link). Informed consent was obtained from each patient.
IHC for GPNMB was performed according to standard procedures using the same antibody described here and a biotin-conjugated donkey anti-goat secondary antibody (Jackson Laboratories, West Grove, PA, USA) 19 (link). Goat IgG, a polyclonal isotype control (Abcam), was used as a negative control. For preliminary experiments, we created cell blocks containing three cell lines (GPNMB expression levels: high, SK-BR-3; moderate, MKN1; and low, LS174T). We used PROTEASE 1 (Ventana, Tuscon, AZ, USA) for antigen retrieval and determined the best conditions for GPNMB IHC (Fig.S1 ). Changes in GPNMB-staining intensity between each primary tumor and metastasis were determined by an experienced pathologist. This study was approved by the Central Ethics Committee of Gifu University.
IHC for GPNMB was performed according to standard procedures using the same antibody described here and a biotin-conjugated donkey anti-goat secondary antibody (Jackson Laboratories, West Grove, PA, USA) 19 (link). Goat IgG, a polyclonal isotype control (Abcam), was used as a negative control. For preliminary experiments, we created cell blocks containing three cell lines (GPNMB expression levels: high, SK-BR-3; moderate, MKN1; and low, LS174T). We used PROTEASE 1 (Ventana, Tuscon, AZ, USA) for antigen retrieval and determined the best conditions for GPNMB IHC (Fig.
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