Antibody diluent

ReNcell CX® and A549 cells were fixed with Roti-Histofix 4% (Roth, Karlsruhe, Germany, #P087.4) (6 min, RT), then washed three times with PBS. Cells were treated 1 h at RT with blocking solution (Zytomed, Berlin, Germany, #ZUC007-100) then incubated with respective primary antibodies listed in Table 4 and incubated at 4 °C overnight.
Subsequently, cell culture slides were washed three times with PBS following incubation with secondary antibody (1 h, RT) (Table 2). All antibody-dilutions were prepared with Antibody-Diluent (Zytomed Berlin, Germany, #ZUC025-100).
Following incubation with secondary antibody, cells were washed three times with PBS, coverslip was separated from each cell culture dish, mounted with Vectashield HardSet and treated with DAPI (Biozol, Eching, Germany #VEC-H-1500). Slides were dried overnight at 4°C, then captured by fluorescence microscopy (Zeiss, Oberkochen, Germany: Zeiss Axio Observer.Z1). Images were analyzed with Image J Software and Corel Draw X7 Software.

Formalin-fixed paraffin-embedded sections (4 μm) of omentum samples or co-cultures gels were heated for 1 hr at 60°C and then submerged twice in xylene for 5 min. Immunohistochemistry was performed using a Vectastain ABC Kit (Elite), as per the manufacturer's instructions. Slides were rehydrated and heated-antigen retrieval was performed using citric acid-based antigen unmasking solution (Vector Laboratories) and heated in a 2100 antigen-retriever (Aptum Biologics). Endogenous peroxidase activity was blocked using 3% H2O2 in PBS and sections were then blocked with 5% BSA. The primary antibody was added in antibody diluent (Zytomed) and covered at room temperature for 1 hr. Slides were incubated with a biotinylated secondary antibody (Vector) for 30 minutes. Subsequent steps were carried out according to the protocol included with the Vectastain Elite ABC HRP kit, after which the slides were incubated with DAB solution made using Sigmafast DAB tablets (Sigma-Aldrich). Finally, slides were counterstained in 50% Gill’s hematoxylin I, washed with water and dehydrated. After leaving to dry, coverslips were affixed using a drop of DPX mountant (Sigma-Aldrich).

Immunohistochemistry was used to determine the intracellular localization and expression of HSP27 and pHSP27 (Ser-15, Ser-78 and Ser-82). Immunohistochemical staining was performed using paraffin-embedded tissue. Tissue sections (4 µm) were deparaffinized in xylene and rehydrated in a descending alcohol set followed by heated antigen retrieval with 10 mM sodium citrate buffer (pH 6.0) for five minutes. Coverplates™ (ThermoFisher Scientific) were used. Endogenous peroxidase activity was quenched with Peroxide Block (Zytomed Systems). Primary anti-HSP27 monoclonal antibody (working dilution 1:500, Abcam (UK), ab2790) and anti-phosphorylated-HSP27-antibodies (Abcam (UK), Ser15: working dilution 1:350, ab76313; Ser78: working dilution 1:900, ab32501; Ser82: working dilution 1:700, ab90537) were diluted with Antibody Diluent (Zytomed Systems). Sections were covered with antibody and incubated at 4 °C for 24 h. Subsequently, ZytoChem Plus (HRP) Polymer Bulk Kit (Zytomed Systems) were used before staining with DAB (diaminobenzidine) Substrate Kit (Zytomed Systems). Gill’s hematoxylin III (Carl Roth) was used as a counterstaining agent, including a 10 s hydrochloric acid bath (5%) for differentiation. Sections were then dehydrated and mounted with EcoMount (Zytomed Systems).

Human macrophages were stained with anti-CD68 (clone PG-M1, DakoCytomation) and anti-CD206 (MRC) (R&D Systems), both diluted 1:100 in Antibody Diluent (Zytomed Systems). The signal was detected using Alexa fluor 488 and 594 conjugated secondary antibodies (Molecular Probes), and the samples were imaged with an Axioskop 2 mot plus fluorescence microscope equipped with Plan-APOCHROMAT 20×/0.8 NA and 40×/0.95 NA objectives and an AxioCam MRc camera with AxioVision software 4.7.1 (Carl Zeiss AG).

Human AECII were seeded on cover-slips, stimulated for 48 h and subsequently fixed for 10 min. With 4% PFA. The PFA was removed and the cells were permeabilized with 0.1% Triton X-100 in PBS for 7 min at room-temperature (RT). After washing with PBS, a blocking step with 1% BSA/PBS was conducted for 20 min. to prevent unspecific binding followed by thorough washing with PBS. The actin-cytoskeleton was visualized by staining with 165 nM TRITC-conjugated phalloidin (Invitrogen, Carlsbad, CA, USA) and 1 μg/ml DAPI (Invitrogen, Carlsbad, CA, USA) diluted in antibody diluent (Zytomed Systems, Berlin, Germany) for 20 min. at RT. The solution was discarded and the cells washed with PBS prior to mounting on cover slides with DABCO (2.5% 1.4-diazabicyclo[2.2.2]octane in 90% glycerol). Images were taken at 200× magnification on a Nikon Eclipse 80i fluorescence microscope equipped with a CCD-camera. The actin signal per cell was quantified using ImageJ software with RGB modus. The specific actin signal was determined as the area of signal in the red channel and divided by number of nuclei (actin area/cell) in the image. All images were acquired at the same exposure time.

Gels were fixed in 10% neutral buffered formalin. Subsequently, Triton X-100 (0.5% in PBS) was added to the gels and incubated for 10 min. Gels were washed and incubated in blocking solution (PBS with 5% BSA), which was replaced with the primary antibody in antibody diluent (Zytomed) and incubated overnight at 4°C. Gels were then washed before a fluorescent secondary antibody was added in antibody diluent and protected from light while shaking for 1 hr. Finally, gels were incubated with PBS containing 0.4 μg/ml DAPI, washed in PBS for 5 min, and imaged using a Zeiss LSM510 confocal microscope using a 10x or 20x air objective. Tetra- and penta- culture images were acquired using a field of view equal to 238.1 x 238.1μm containing 1024x1024 pixels. All imaging conditions were kept constant for all the experiments.

Fixation of cells was conducted as described above. Permeabilization was conducted with 0.25% Triton X-100/PBS for 7 min at RT. Double staining of target antigens was achieved by incubation with first primary antibody for 45 min. Followed by subsequent washing with PBS and incubation with the second primary antibody at the same conditions. Visualization of primary antibodies was conducted with Alexa488 or TRITC labeled secondary antibodies (Molecular Probes, Eugene, Oregon, USA) diluted at 1/200 with 1 μg/ml DAPI in antibody diluent (Zytomed Systems, Berlin, Germany) for 45 min. at room temperature. Unbound antibodies and DAPI were removed by washing with PBS before mounting of cover slips on microscopic slides (Super Frost Plus, Menzel Gläser, Germany) with DABCO. In general, each reagent step was followed by thorough washing with 200 μl of PBS. To detect cells of hAECII lineage, the surfactant protein transcription factor TTF-1 [19 (link)] was targeted with mouse anti-TTF-1 (clone SPT24, Zytomed Systems, Berlin, Germany) at 1/400 dilution. Activity of collagen production and secretion processes were assessed by targeting the collagen chaperon HSP47 [20 (link)] with rabbit anti-HSP47 (clone EPR4217, Abcam, Oxford, UK) at 1/100 dilution.