Cryosections (7 μm thick) were labelled without prior fixation using primary antibodies diluted in PBS 0.1% Tween-20. Immunofluorescence was visualised with species-specific secondary antibodies directly linked to Alexa-fluorophores (Molecular Probes, Eugene, OR, USA). Nuclei were visualised using DAPI (Molecular Probes, Eugene, OR, USA) diluted in PBS 0.1% Tween-20. Immunohistochemistry was performed using Novo-Link peroxidase detection kit according to manufacturer’s instructions (Novo-Link peroxidase detection kit, Leica Microsystems, Wetzlar, Germany). We used mouse monoclonal antibody against MHC Class I (Dako, Glostrup Denmark, Cat. M0736, 1:1000) and rabbit polyclonal antibody against active caspase-3 (Pharmingen, San Diego, CA, USA, Cat. 559565, 1:400). Non-specific labelling was assessed using sections incubated without primary antibodies. Immunofluorescence was visualised under a conventional epifluorescence microscope (Leica DM5000B). Images were acquired with Leica Imaging Suite (Leica Microsystems, Wetzlar, Germany) and were quantified with ImageJ software (http://rsbweb.nih.gov/ij/ ).
Novolink peroxidase detection kit
Manufactured by Leica
Sourced in Germany, United Kingdom
The Novolink® peroxidase detection kit is a laboratory reagent used in the visualization of specific target proteins or antigens in tissue samples during immunohistochemistry (IHC) procedures. The kit contains the necessary components to amplify and detect peroxidase-based signals, allowing for the identification and localization of target biomolecules in the samples.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluorophore, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Dm5000b, by Leica (1 mentions)
Pierce co immunoprecipitation co ip kit, by Thermo Fisher Scientific (1 mentions)
Vector novared substrate kit, by Vector Laboratories (1 mentions)
Vector novared, by Vector Laboratories (1 mentions)
3 protocols using novolink peroxidase detection kit
1
Immunofluorescence and Immunohistochemistry of Cryosections
2
ErbB Receptor Dimerisation Analysis
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To determine ErbB dimerisation, co-immunprecipitation of ErbB receptor complexes was performed according to the manufacturer's protocol (Pierce co-immunoprecipitation (Co-IP) kit, ThermoFisher Scientific, UK). This is discussed in detail in Appendix S1 1 and the antibody list detailed in Table SIII 1 . Blots were developed using nitro blue tetrazolium/S-bromo-4-chloroindoxyl phosphate (NBT/BCIP, 34042, ThermoFisher Scientific, UK). Immunostaining FFPE sections (5 µm) were stained for ErbB2 using manufacturer's instructions for Novolink ® peroxidase detection kit (Leica Biosystems, UK) and red chromogen Vector ® NovaRED™ substrate kit (Vector, CA, USA) to differentiate from melanin then counterstained with haematoxylin. Combined ErbB2/NRG1 immunofluorescence was performed using citrate buffer (pH 6) antigen retrieval for 20 min at 90°C. A negative control was performed in each without primary antibody. (Antibody incubation details Table SIII 1 ).
3
AKR1B10 and CRABP2 Expression in Keloid Tissues
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AKR1B10 tissue detection was performed using the Novolink peroxidase detection kit (Leica Biosystems, Milton Keynes, UK) and red chromogen counterstaining (Vector NovaRED, Vector Laboratories, Peterborough, UK). Combined AKR1B10/CRABP2 immunofluorescent staining was performed on keloid and normal skin tissue. AKR1B10 immunocytochemistry was done on transfected NHEK to confirm plasmid incorporation and protein expression. Further methodology is described in the Supplementary Materials and Methods and the antibody list in Supplementary Table S4a online.
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