Antigen retrieval

Antigen retrieval (Dako) and endogenous peroxidases blocking (3% H₂O₂) were performed before free-floating staining. After washing in washing buffer (0.3% Triton X-100 in 0.05M Tris-buffered saline), the sections were incubated with 1% bovine serum albumin in washing buffer for an hour, before overnight incubation of primary antibody rabbit anti-CD207 (1:500, Invitrogen). Sections were washed in washing buffer and then incubated with secondary antibody goat antirabbit-HRP (1:400, Dako) at room temperature for 2 hours. After rinsing sections with 0.3% TBST buffer, sections were treated with DAB working solution (0.05% DAK, 0.1% H₂O₂) for 5 minutes. Sections were rinsed in washing buffer and mounted on slides with gelatine. Skin sections were air dried on slides and rehydrated in distilled water and dehydrated in 70% ethanol (10 minutes), 90% ethanol (10 minutes), 99% ethanol (15 minutes), and xylene (15 minutes) and finally coverslipped.

Cryosections were air‐dried for 30 minutes and hydrated in Tris‐buffered saline (TBS; pH 7.6) for 30 minutes. Slices were treated with Antigen Retrieval (Dako, Les Ulis, France) for 15 minutes at 95–96°C and then cooled to room temperature during 20 minutes. Nonspecific binding was blocked by incubation in TBS + bovine serum albumin (BSA) 1% + normal goat serum (10%, Gibco/Life Technologies, Grand Island, NY) for 30 minutes. The primary antibody was incubated overnight in TBS + 1% BSA at 4°C for 12 hours. After washing in TBS, fluorophore‐conjugated secondary antibodies were coincubated in Dako Diluent (Dako) for 1 hour at room temperature. After washing in TBS, sections were stained with 4′,6 diamidino‐2‐phenylindole (DAPI; Molecular Probes, Eugene, OR; 1:4,000 in TBS) for 10 minutes at room temperature. Sections were mounted in Fluoromount G (SouthernBiotech, Birmingham, AL).

Immunohistochemistry was performed on the retinal paraffin sections. The sections were de-deparaffinized and hydrated. After pretreatment with antigen retrieval (DAKO) at 95°C for 15 min, the sections were left to cool for 15 min at room temperature. Endogenous peroxidase activity was inhibited by incubating the slides in 0.3% H₂O₂ for 30 min. The sections were then incubated overnight at 4°C with the primary antibodies. Caspase-3 rabbit polyclonal antibody (Lab Visions Corporation Laboratories, catalog number PA1-26426) was used at a concentration of 1:100. The secondary anti-rabbit antibody detection system (Envisions, Dako) was used for 20 min at room temperature. Finally, diaminobenzidine 0.05% (Dakopatts, Glostrup, Denmark) was applied as a chromogen at room temperature for 3 min. Mayer’s hematoxylin was used as a counterstain. Lastly, dehydration, clearing, and mounting were done. Tonsil was used as a positive control slide. Negative control slides were prepared by replacement of the primary antibodies with phosphate-buffered saline.^{29 (link)}

Staining of formalin-fixed, paraffin-embedded breast cancer samples (139 cases that have received chemotherapy) was performed as previously described (Ghebeh et al, 2006 (link)). Briefly, after deparaffinization and rehydration, antigen retrieval (Dako) was used before blocking endogenous peroxidase for 15 min with 3% hydrogen peroxide (Sigma) in methanol. Sections were then blocked with 10% goat serum (Sigma) for 60 min, followed by the addition of a primary anti-human fascin antibody (1/200) overnight at 4 °C. After washing, sections were incubated with labelled Polymer (EnVision⁺ anti-mouse) HRP detection kit (Dako) for 30 min at room temperature. The HRP was detected using DAB substrate (Novocastra, Buffalo Grove, IL, USA) for 4 min and the sections were counterstained for 1 min with Harris hematoxylin (Acros Organics, Pittsburgh, PA, USA). Slides were washed with distilled water, dried and cover-slipped using permanent mount (Novocastra). The intensity of staining and the percentages of fascin-positive cells were evaluated by two independent pathologists who had no prior knowledge of patient details.

The kidney tissue sections (4 μm in thickness) were prepared. After deparaffinization and antigen retrieval (Dako, CA, USA), the sections were then blocked and incubated with antibody against ACSL4 (Abcam, Cambridge, UK, 1:500) for 1 h followed by incubation with an appropriate secondary antibody (Abcam, 1:1000) for 1 h. The sections were stained with DAB and then counterstained, dehydrated, and mounted. The images were taken using a microscope. Images quantified by Image‐Pro Plus 6.0 analysis software.