3 3 diaminobenzidine (dab)

For immunohistochemistry (IHC) on murine tissues, lung sections were deparaffinized and rehydrated, and then subjected to heat-mediated antigen retrieval with 10 mM sodium citrate buffer (pH = 6.0) at 95°C for 15 min. After blocking of endogenous peroxidase activity with 3% hydrogen peroxide (15 min, RT), sections were blocked with 10% normal goat serum (1 h, RT) followed by blocking of endogenous biotin using an Avidin/Biotin blocking kit (Vector Laboratories, Burlingame, CA, United States). Afterwards, primary antibodies for F4/80 (rat anti-mouse F4/80, clone Cl:A3-1, 1:100, AbD Serotec; Kidlington, United Kingdom), and FR-β (rabbit anti-mouse FR-β, 1:400, Genetex, Irvine, CA, United States) were applied on the specimens and incubated overnight at 4°C. Isotype- and concentration-matched IgGs served as negative controls. Next, biotin-labeled goat anti-rat or anti-rabbit secondary antibodies (all from Vector Laboratories) were applied (30 min, RT). This was followed by incubation with the Vectastain ABC Elite HRP kit for 30 min at RT (Vector Laboratories). Finally, stainings were visualized using 3,3′-diaminobenzidine (DAB) in case of F4/80, or 3-amino-9-ethylcarbazole (AEC) (all from Vector Laboratories) in case of FR-β, and sections were counterstained with Mayer's hematoxylin (J.T. Baker, Deventer, Netherlands).

Immunohistochemistry (IHC) was performed as previously described^{17 (link)}. For peroxidase IHC analysis, sections were incubated with the primary antibody overnight at 4 °C, followed by incubation with biotinylated anti-mouse (Vector Laboratories, Burlingame, CA, USA) or biotinylated anti-rabbit (Vector Laboratories) secondary antibodies at RT for 1 h. The samples were incubated in the avidin-biotin complex solution (Vector Laboratories), they were then stained with 3, 3′ diaminobenzidine (DAB; Vector Laboratories) for 5 min and counterstained with Mayer’s hematoxylin (Muto, Tokyo, Japan). The signals were analyzed using a bright field microscope (Nikon TE-2000U). The fluorescence signal was visualized using a confocal microscope (LSM510; Carl Zeiss, Oberkochen, Germany) at excitation wavelengths of 488 nm (Alexa Fluor^® 488), 543 nm (Alexa Fluor^® 555), and 405 nm (DAPI). At least three fields of view per section were analyzed. The IHC images were quantified from the representing images using an IHC profiler in the ImageJ software.

Immunohistochemistry was performed using polyclonal antibody for SphK1 and COX-2. Tissue samples were recovered, fixed in 10% buffered formalin, and embedded in paraffin. Section (4 µm) were dewaxed and hydrated through graded ethanol, cooked in 25 mM citrate buffer, pH 6.0, in a pressure cooker for 10 min, transferred into boiling deionized water, and let to cool for 20 min. Tissue sections were then treated with 3% hydrogen peroxide to inactivate endogenous peroxidase activity. The slides were incubated with anti-SphK1 (Abcam) and anti-COX-2 (Cell Signaling) antibodies at a working dilution of 1:200 overnight at 4 °C [18 (link)] and a secondary polymer-based detection system (EnVision+ System Labelled Polymer-horseradish peroxidase anti-rabbit; Dako). The chromogen was 3,3′diaminobenzidine (DAB; Vector Laboratories, Burlingame, CA, USA) and sections were counterstained with haematoxylin. The specificity of technique was evaluated by negative controls (omitting the incubation with the primary antibody and incubating it with nonimmune sera). Areas of staining were analyzed by WinRoof version 6.3 (Mitani, Tokio, Japan) software with ten non-overlapping randomly chosen histological fields. Results were expressed as the percentage of stained cells in each field.

Tissues were fixed by immersion in 10% formalin and paraffin-embedded according to standard procedures. Two 4 μm-thick serial sections were cut and mounted on poly-L-lysine-coated glass slides. Sections were deparaffinized and underwent antigen retrieval by microwave oven treatment (5 min × 3 times, in 1% sodium citrate buffer, pH 6.0). Non-specific bindings were blocked by incubation (30 minutes at room temperature) with 1.5 % non-immune mouse serum (Dako). Endogenous peroxidases were quenched with 0.3% hydrogen peroxide in methanol. Slides were rinsed twice with Tris-HCl buffer and incubated 1 hour at room temperature with anti-MAPK15 antibody. For negative controls, non-immune serum in TBS buffer (1:500) was used instead of the primary antibody.
The standard streptavidin-biotin linked horseradish peroxidase (LSAB) technique was then performed with the Dako LSAB kit HRP (Dako). 3,3′-diaminobenzidine (DAB; Vector Laboratories, Burlingame, CA) was used as chromogenic substrate. Sections were counterstained with Mayer's haematoxylin for 30s, mounted and cover-slipped with a synthetic medium.
The immunohistochemical expression was evaluated semiquantitatively and cells were classified as low staining, [from 0 (<5%), to + (5-25%)], and high staining [from ++ (26-50%) to +++ (>50%)].

IHC staining of lung tumor tissue was performed using rabbit anti-human active caveolin-1 antibodies as described earlier [28 (link), 29 (link)] on the mouse model tumors and on 20 de-identified human lung carcinomas provided by the University of Maryland School of Medicine Center for Innovative Biomedical Resources Pathology Biorepository Shared Service, Maryland after an IRB approval from University of Maryland School of Medicine. Briefly, the lung tumors tissues were embedded in paraffin and sectioned at 2-4-μm thickness. Staining was performed using the kit (Abcam, MA, USA) following the manufacturer’s standard protocol (Vector Laboratories, Burlingame, CA). The tissue slides were blocked with 2.5% normal horse serum for 10 min. Samples were then incubated with rabbit anti-human active caveolin-1 antibody (dilution 1:50), for 12 hours at 4°C. Following thorough wash, the tissue slides were incubated with anti-rabbit IgG HRP secondary antibody for 10 minutes. The slides were then stained with 3,3′-diaminobenzidine (DAB) (Vector Laboratories) and counterstained with hematoxylin (Vector Laboratories), dehydrated, treated with xylene, and mounted. All slides were examined, pictures were taken using an Olympus BX41 microscope (Olympus America, Melville, NY).