Power block

IMCD cells (5000 cells/well) were seeded on BD multichamber slides and underwent the gradual increase in osmolarity as described above. At 330, 600 and 900 mOsm, cells were washed with ice-cold PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. After washing with PBS, cells were blocked using 1× Powerblock (Biogenex, San Ramon, CA, USA) for 10 min and incubated with rabbit polyclonal TGF-ß1 antibody (1:50 in PBS, Santa Cruz) overnight at 4C. The slide was carefully washed twice with PBS and incubated with secondary antibody (Rabbit Link, Biogenex) for 30 min, washed and developed with Fast Red (Dako). Nuclei were counterstained with hematoxylin solution, then slides were mounted using Aquatex (Merck) and analyzed under light microscope.

Tissue sections were deparaffinized in xylene and hydrated with ethanol gradations and water. Ly6G, CD20 and CD3 epitopes were retrieved by the heat-induced antigen retrieval method using 10 mM citrate buffer (pH 6). Endogenous peroxidase activity was blocked using 0.3% hydrogen peroxide and then subsequently blocked with 1× Power Block (BioGenex). Sections were then incubated with primary goat anti-mouse IgG CD20 antibody (clone M-20, Santa Cruz Biotechnology), rat anti-mouse IgG Ly6G antibody (clone 1A8, BioLegend) and rat anti-mouse IgG CD3 antibody (clone 17A2, BioLegend) at a 1:100 dilution overnight at 4°C in Power Block. Sections were then washed with PBS-0.05% Tween 20 and incubated with biotinylated secondary antibodies (goat anti-rat IgG, BD 559286; rabbit anti-goat IgG, BA-5000, Vector Laboratories) at a 1:100 dilution for 45 min. Streptavidin horseradish peroxidase (BioGenex, HK3305K) was used to label the secondary antibody for immunodetection by DAB chromogen (Biogenex). After counterstaining with Mayer's Hematoxylin (BioGenex), the samples were dehydrated with ethanol gradations, dipped in xylene and mounted using Cytoseal (Thermo Scientific).

Frozen samples were sectioned at 10 μm using CryoJane (Leica Microsystems). The slides were blocked in 1× Power Block (BioGenex) in PBS at room temperature for 1 h, then stained with primary antibody diluted in blocking buffer overnight at 4 °C. The next day, the slides were washed in PBS 3 × 5 min, incubated in secondary antibody in blocking buffer for 1 h at room temperature, and washed in PBS 3 × 5 min before mounting with DAPI (Vectashield). The primary antibodies used were goat anti-GFP (FITC) (1:200; abcam) and rabbit anti-beta galactosidase (abcam). Anti-Rabbit-AlexaFluor488 (Life Technologies) was used for the secondary antibody for immunofluorescent staining.