Eclipse ti2 e fluorescence microscope

Formalin-fixed, paraffin embedded tissues were sectioned, baked at 60°C for 30 min, dewaxed in xylene and rehydrated through an ethanol gradient per standard procedures, and then subject to antigen retrieval with citrate buffer (pH 6.0) in a pressure cooker. Tissues were washed in PBS-T (PBS +0.05% Triton X-100), blocked with 2% BSA in PBS-T, and then incubated overnight with 1:1000 anti-SDMA produced as previously described.⁴² (link) Tissues were washed, stained with anti-rabbit HRP (Cell Signaling), and Tyramide signal amplification (TSA) was performed with CF550R Dye (Biotium, #96077) in amplification buffer (Akoya Biosciences). Slides were subject to additional antigen retrieval in citrate buffer, and then the procedure repeated with anti-pan cytokeratin (clone AE1/3, Bethyl), anti-mouse HRP (Cell Signaling), and CF680R (Biotium, #92196). Finally, slides were mounted using Vectashield with DAPI (Vector Labs) and imaged using a Nikon Eclipse Ti2-e Fluorescence microscope. At least 5 images per tissue were captured and quantified using a custom MATLAB algorithm (R2020a, Mathworks). SDMA is reported as average SDMA intensity in the tissue after subtracting background intensity determined from a control with no primary antibody. All images per tissue were averaged to determine the SDMA intensity in that tissue.

Paraffin-embedded tissues were cut into 2.5 μm-thick sections, deparaffinized, and dehydrated in a graded ethanol series. Masson’s trichrome staining was performed according to the manufacturer’s instructions. According to the manufacturer’s protocols, Immunohistochemistry (IHC) was performed using the UltraSensitiveTM SP IHC Kit and DAB reagent kit. Briefly, the sections were subjected to antigen retrieval, removal of endogenous peroxidase activity, and antigen blocking. Sections were subsequently incubated overnight with a primary antibody (CTSG, 1:400; collagen I, 1:400) at 4 °C, followed by incubation with a horseradish peroxidase-labeled secondary antibody. DAB was used for color visualization. The sections were counterstained with hematoxylin. The following primary antibodies were used for double-immunofluorescence staining of sections: rabbit anti-CTSG antibody (1:400) and SMA-α-FITC (1:500). Alexa Fluor 555 anti-rabbit antibody was used as the secondary antibody. We used 4′,6-diamidino-2-phenylindole (DAPI) for counterstaining. Pictures were recorded using a Nikon Eclipse Ti2-E fluorescence microscope. Images (× 400) from each section were analyzed blindly and quantified using ImageJ software.

For organoid staining, cells were plated in 5% Growth Factor Reduced Matrigel Matrix (Corning) and 3 to 5 d later were harvested in cold PBS. Organoids were then washed with 1X Carbo-Free Blocking Solution (Vector Laboratories) for 5 min and stained using biotin-StcE^E447D (34 (link)) in PBS for 1 h on ice. After a wash with PBS, cells were stained with Streptavidin-647 (Life Technologies, 1:1,000) for 20 min at room temperature and washed again with PBS. Cells were then resuspended in a drop of Vectashield (Vector Laboratories) to mount on glass slides and image right away using the Nikon ECLIPSE Ti2-E fluorescence microscope. Quantifications were done using imageJ where MFI was calculated for manually surrounded organoids. For BMSC staining, cells were plated on collagen-coated glass slides, fixed with 4% PFA, permeabilized with 0.1% Triton-X 100 in PBS and blocked with 1% BSA-PBS before incubation with primary antibodies [anti-PDGFRβ (BioRad 7460-3104, 1:200) and anti-αSMA (Fisher, MS113P, 1:500)] overnight. After washes with PBS, slides were incubated with secondary antibody for 30 min (Life Technologies, Alexa Fluor 647 anti-Mouse IgG2a, Alexa Fluor 488 anti-Mouse IgG1, 1:500) and imaged as described.