Immunohistochemistry was used to confirm pepsin protein presence in BE and absence in neighboring normal tissue. Esophageal biopsies were fixed in formalin, paraffin-embedded, sectioned to 4um and mounted to glass slides. Following deparaffinizing, antigen retrieval was performed on PT Link (Dako) at 97°C for 20 minutes. Immunohistochemistry with mouse anti-pepsin antibody, peroxidase-conjugated secondary antibody (Dako), diaminobenzidine, and hematoxylin was performed on the Autostainer Plus using the EnVision™ FLEX High pH Detection Kit (Dako). Images were collected on a Nikon Eclipse Ti using NIS Elements software (Nikon, Tokyo, Japan).
Envision flex high ph detection kit
Manufactured by Agilent Technologies
The EnVision™ FLEX High pH Detection Kit is a laboratory equipment product designed for immunohistochemistry (IHC) applications. It provides a method for the detection of target antigens in tissue sections. The kit includes reagents and solutions necessary for the amplification and visualization of the target signal.
Automatically generated - may contain errors
3 protocols using envision flex high ph detection kit
1
Immunohistochemical Pepsin Detection in BE
2
Immunohistochemical Staining of Acid Ceramidase
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All immunohistochemical (IHC) staining was performed on a Dako Autostainer Plus using the Dako Envision™ FLEX High pH detection kit. Briefly, after deparaffinization and rehydration of the tissue, antigen retrieval was performed with Tris/EDTA pH 9. After blocking of non-target epitopes, anti-acid ceramidase primary antibody (Santa Cruz Biotechnology Inc., Dallas, TX, USA) was applied at a concentration of 1:100 for 30 min, secondary antibody for 20 min, and DAB for 10 min. Hematoxylin was used as counterstain.
3
Evaluating BRAFV600E and VEGFR Expression in BHT-101 Tumors
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Ex vivo immunohistochemistry was performed to evaluate changes in morphology as well as BRAFV600E and VEGFR expression in BHT-101 tumors. Mice bearing BHT-101 tumors were treated with onalespib and/or sorafenib as previously described (n = 12). When tumors reached a size of 1000 mm3 or day 25 (endpoint of experiment), tumors were collected and fixed in 4% buffered formalin and transferred to 70% ethanol. Tumor tissues were paraffin embedded, sectioned, and deparaffinized. Immunohistochemical stainings were performed on FFPE sections using fully automated protocols (DAKO Autostainer Link48) and Envision FLEX, high pH detection kit from DAKO #K8000. The following antibodies were used: BRAF V600E (Sigma Aldrich, #SAB560047, rabbit monoclonal, clone RM8, diluted 1:2000) and VEGFR3 (abcam, #ab27278, polyclonal, diluted 1:400). Immunohistochemical sections were manually scored according to staining intensity (negative −, weak +, moderate ++, or strong +++).
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