High ph antigen retrieval solution

Formalin-fixed paraffin-embedded tissue blocks were prepared from surgical specimens, and sections were stained with hematoxylin and eosin for histopathological examination by two gastrointestinal pathologists. Immunohistochemical staining for MLH1 was performed as described previously [36 (link)]. In brief, 4µM sections were cut, dewaxed and rehydrated. High pH antigen retrieval solution (pH 9.0; Dako, Glostrup, Denmark) was used for MLH1 at 112°C for 7 min. All sections were stained using the MLH1 antibody (clone G168-15, 1:100; BD Pharmingen). Loss of MLH1 expression was recorded when nuclear staining was observed in normal tissue but not in adjacent malignant cells.

Formalin fixed paraffin embedded tissue was sectioned at 4-microns and dried overnight at 58°C. Slides were deparaffinized and hydrated stepwise followed by heat-induced epitope retrieval (HIER) in high pH antigen retrieval solution (Dako) at 110°C for 20 minutes using a Biocare Medical Decloaking Chamber. Slides were incubated in Galectin-1 antibody (R&D AF-1152) diluted at 1:50 for one hour at room temperature followed by incubation with polymer bound anti-goat secondary for 30 minutes (Vector Laboratories Immpress kit). Staining was visualized by incubation with 3, 3′-diaminobenzidine (DAB) chromogen (Dako, CA) for 5 minutes and slides were lightly counterstained with hematoxylin.

Six cases of triple negative breast cancer from formalin fixed surgical archives of the University of Kentucky were selected and 4-micron thick sections were cut from each and dried overnight at 58°C. Slides were deparaffinized and hydrated stepwise followed by heat-induced epitope retrieval (HIER) in high pH antigen retrieval solution (Dako) at 110°C for 20 minutes using a Biocare Medical Decloaking Chamber. Slides were incubated in Galectin-1 antibody (Sigma #HPA000646) diluted at 1:400 for 30 minutes at room temperature followed by incubation with polymer bound anti-rabbit secondary for 30 minutes (Dako Envision+). Staining was visualized by incubation with 3, 3′-diaminobenzidine (DAB) chromogen (Dako, CA) for 3 minutes and slides were lightly counterstained with hematoxylin.